Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution

J. Mol. Biol. (1989) 208, 679-696

Crystal Structure of the p-Hydroxybenzoate HydroxylaseSubstrate Complex Refined at 1.9 A Resolution Analysis of the Enzyme-Substrate and Enzyme-Product

Complexes

Herman A. Schreuderl?, Peter A. J. PrickI, Rik K. Wierengal Gerrit Vriend’, Keith S. Wilson2, Wim G. J. Hoi’ and Jan Drenth’ 1Laboratory of Chemical Physics, University of Groningen NCjenborgh 16, 9747 AG Groningen, The Netherlands 2EMBL Outstation, Notkestrasse 85 2000 Hamburg 52, F.R.G. (Received 24 December 1988, and in revised form 21 April

1988)

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxybenzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 156% for 31,148 reflections between 6.0 and l-9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7 yc for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14” compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10” for the enzyme-substrate complex and 19” for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring. This arrangement may stabilize negatively charged forms of the isoalloxazine ring that occur during catalysis. Two solvent molecules in the active site are in van der Waals’ contact with the flavin ring. Of particular interest is the position of a solvent molecule near the N terminus of helix HlO. This solvent molecule is within hydrogen-bonding distance of two peptide nitrogen atoms and could be replaced by the distal oxygen atom of the flavin 4a-peroxide anion after reaction with oxygen.

t Present

address:

0022-2836/89/160679-18

Molecular $03.00/O

Biology

Institute,

405 Hilgard 679

Avenue,

Los Angeles,

CA 90024, U.S.A. 0 1989 Academic Press Limited

H. A. Schreuder

680

1. Introduction p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoenzyme that catalyses the conversion of p-hydroxybenzoate to 3,4-dihydroxybenzoate using molecular oxygen and NADPH according to the following reaction:

p

+ O2 + NCDPH

cl-

+ H’

-

HO

I,0

C, + Hz0 0‘

+ NADP’

The reaction is part of the P-ketoadipic acid pathway, which is found in several pseudomonad strains (Dagley, 1982). Via this pathway, these organisms degrade many aromatic compounds, which are produced in nature in bulk quantities by biodegradation of lignin. Lignin is one of the principal components of wood and, after cellulose, the most abundant biopolymer. p-Hydroxybenzoate hydroxylase from Pseudomonas jluorescens has been studied intensively. The enzyme has 394 amino acid residues and its amino acid sequence has been elucidated by Beintema and co-workers (Hofsteenge et al., 1983; Weijer et al.. 1983; Wijnands et al., 1986). Miiller and coworkers studied the enzyme by chemical modification (Wijnands et al., 1987) and with magnetic resonance techniques nuclear (Vervoort, 1986). Detailed kinetic investigations by Massey and coworkers (Entsch et al., 1976; Husain et al., 1980) and work by many others revealed that several subsequent steps are involved in the enzymic reaction of p-hydroxybenzoate hydroxylase: (1) Binding of substrate by the holoenzyme. (2) Reduction of enzyme-bound FAD by NADPH. (3) Reaction of the reduced flavin with molecular oxygen to form flavin 4a-hydroperoxide. (4) Hydroxylation of the substrat,e by the flavin 4a-hydroperoxide. (5) Release of the product’. Crystallographic investigat)ions in our laboratory have yielded the crystal structures of several the reaction path of intermediates along p-hydroxybenzoate hydroxylase (Wierenga et al., 1979; Van der Laan, 1986; Schreuder et al., 1987). In order to compare the differences between the crystal structures reliably, they must be of high accuracy For this reason, it was important to determi& the structure of at least one of the p-hydroxybenzoate hydroxylase complexes at high resolution. Although crystals of the p-hydroxybenzoate hydroxylase substrate complex (also denoted as PHBH . FAD. pOHB) do not diffract much beyond 2.3 a using conventional X-ray sources. it was possible to collect data up to at least 1.9a (1 A=O.l nm) using synchrotron radiation. This paper describes the structure of p-hydroxybenzoate hydroxylase with bound substrate. refined at a resolution of 1.9 A. This model is analysed. with particular regard to active site geometry and

et al

FAD binding, and is compared with the 2.3 A structure of the p-hydroxybenzoate hydroxylaseproduct complex.

2. Materials and Methods (a) Purification

and crystallization

A detailed description of the purification of p-hydroxybenzoate hydroxylase from Pa. jluorescens has been published by Van der Laan et al. (1989). The final DEAE chromatography step used in this procedure was essential to remove microheterogeneity of p-hydroxybenzoatr hydroxylase. which is due to partial oxidation of Cysll6 (Van der Laan, 1986: Van Berkel & Miiller. 1987; Van der Laan et al., 1989). The protein was crystallized with the free interface diffusion method in sealed capillary tubes essentially as described by Drenth et al. (1975) and by Van der Laan et al. (1989): 5 ~1 of a solut’ion containing 5 to 10 mg 0.04 mM-FAD. protein/ml, 2 mM-p-hydroxybenzoate. 0.3 mM-EDTA, 30 mm-sodium sulphite in 0.1 m-phosphate buffer (pH 7.5). was layered on t,op of .5~1 of a 70% saturated ammonium sulphate solution in 0.1 M-phosphate buffer (pH 7.5). The temperature was raised from 4°C to 20°C over a period of 2 weeks, After 2 months. crystal growth was completed. resulting in crystals of the enzyme-subst,rate with complex dimensions up to 0,2 mm x 0.5 mm x 0.8 mm. The spare group was C222, with a= 71.5 A. h = 145.8 .& and c=88.2 !I. (11)Data collection

and processiny

Two datasets were combined for the 1.9 x structure determination of the enzymesubstrate complex. The 1st was collected on film using monochromated (luKa radiation from a rotating anode generator (Wierenga et al., 1979). The 2nd dataset was collected using synchrotron radiation from beam-line Xl 1 of the Hamburg Outstation of the European Molecular Biology Lahoratory. The wavelength was 1.482 A and the crystal-to-film distance was 55.0 mm, giving a resolution of 1.8 A at the edge of the film. Tn order to prevent loss of high resolution reflect,ions due to radiation damage, the “8meriran data collection method” was used (Rossmann 1983). Crystals were aligned by r>-r. & Erickson. oscillation pictures were made and afterwards 2 still photographs were taken to obt,ain the orientation. B total of 4 to 16 exposures with 1.2” of oscillation were taken per crystal. The exposure time per film was 1 t)o 3 min. In total, 13 crystals were exposed. but data from 3 crystals had to be discarded because of large mosaic spread or twinning problems. Films were scanned with a 50 pm raster on a Scandig microdensitometer (,Joyrr -Loebl) equipped with a photomultiplier. A tot,al of 77 filmpacks (A + B films) was processed using the oscillation film package written by $1. (:. Rossmann and co-workers (Rossmann. 197!$: Rossman ut al.. 1979). These programs. as well as t,he refinement in programs, were run on a Cyber 205 romputrr Amsterdam. In order to establish (~11 parameters.

wavelength and crystal-to-film distance. trial runs were done with films from different crystals using options to refine the parameters mentioned. It, appeared t,hat the films could be processed using t,he cell dimensions obtained by Wierenga, rt ol. (1979) from rotating anode data and with the wavelength and cnrystal-to-film distance as measured at the synchrotron.

1.9 A Structure

of p-Hydroxybenzoate

In order to obtain sufficient intensity for the weak high-resolution reflections, relatively long exposure times were needed for the synchrotron films. As a result, the lowresolution reflections were mostly overexposed. Also the background in the region up to 3.0 a was very high. Some of the crystals appeared to be intergrown with a small 2nd crystal, resulting in a weak 2nd diffraction pattern at low resolution. But since a 2.5 A rotating anode dataset was already available and neither the background, nor the weak 2nd diffraction pattern was much of a problem beyond 2.5 A, the films gave valuable high-resolution information. Overexposed reflections and a high background at low-resolution made the A films useful only beyond 3.1 A. After merging the A and the B films together, all films were brought to the same scale and temperature factor with the program SCALEKB from the Groningen BIOMOL protein structure determination software package. Postrefinement and merging of the films were done with programs from the Purdue package. The final Rrnerge was 12.6% (R,,,,=~(lli-fl)/Zf). The synchrotron dataset was scaled to the rotating anode dataset by applying a scale and isotropic B-factor with the program KBRANI from the BIOMOL package. From comparison of the datasets, it appeared that the strong reflections of the synchrotron dataset, especially at low resolution, were systematically too weak. Therefore, all reflections from the synchrotron dataset with a resolution of less than 3.0 A and all strong reflections were rejected. The merging R-factor between the datasets, involving 5009 common reflections between 2.5 A and 3.0 A. is 14.2% (R=2ZjF,-F,I/CIF,

+F,I).

The merged dataset was analysed by plotting the average (FI as a function of resolution and by comparing the F,, values with F, values from the 2.5 A starting model (see section (c), below). Sharply increasing R-factors and decreasing correlation coefficients beyond 1.9 A indicated that data contained useful information only up to 1.9 A. The final dataset was 88% complete t,o this resolution limit. (c) Rejnement of the 1.9 A structure of the enzyme-substrate complex

The starting model of the enzyme-substrate complex was obtained by superimposing the refined model of the PHBH . FAD. 3,4-diOHBt complex having an R-factor of 19.30/b (Schreuder et al., 1988a) on to a model of the PHBH . FAD. pOHB that had an R-factor of 26.6% at 2.5 d (Wierenga & Prick, unpublished results). Only the protein atoms of the superimposed model of the PHBH . FAD. 3,4-diOHB complex were used. The FAD, the p-hydroxybenzoate molecule and the solvent molecules were taken from t,he 2.5 A model of the PHBH FAD.pOHB complex. After 12 cycles of Konnert-Hendrickson restrained least-squares refinement (Hendrickson & Konnert. 1980; Hendrickson. 1985) using the rotating anode data, starting at 3.0 .& and gradually increasing the resolution. a 2.5 A model was obtained with an R-factor of 21.4%. This model was used as a starting point for refinement against, the combined 1.9 /! dataset. Model rebuilding was t Abbreviations used: PHBH, p-hydroxybenzoate hydroxylase: 3.4-diOHB, 3,4-dihydroxybenzoate; pOHB, p-hydroxybenzoate: PHB . FAD. pOHB, enzyme-substrate complex; PHBH . FAD. 3,4-diOHB. enzymeprodurt complex; r.m.s. root-mean-square: Sol. solvent.

Hydroxylase

681

carried out on an Evans & Sutherland PS390 computer graphics system, running FRODO software (Jones, 1985) and using electron density maps with coefficients (2mF,,-DF,) exp (ia,) (Read, 1986). The refinement was completed after 73 cycles of Konneti-Hendrickson restrained least-squares refinement and 8 rounds of manual model revision. Some of the major corrections made were the following. At cycle 8, at a resolution of 2.2 A, the segments 61-63, 191-193, 248-254, 261-263 and the side-chain of Glu323 were omitted from the model because their correct position in the electron density was not clear. At cycle 14 these segments were rebuilt in the electron density, which was calculated with a,-weighted (Read. 1986) (2mF,,- DF,‘) exp (ia,‘) coefficients, where F,.’ and a,’ are calculated without the omitted residues. With this procedure, the model bias due to incorrectly placed residues was minimized. At cycle 41, the same was done for the segments 4344, 79-80, 143-145, 391-394 and the side-chain of Asp355. The C-terminal residues 391 to 394 were not rebuilt in the electron density because they appeared to be disordered. In addition, a total of 330 bound solvent molecules was placed by searching (rA weighted (F,,- F,.) maps for peaks of at least l/3 of the standard deviation of the electron density in the corresponding oA weighted (2F, - F,) map. Peaks too close to the protein, or further than 3.5 A from protein or other solvent molecules, were rejected. Due to the limited resolution of the data, only individual K-factors were refined and all occupancies of solvent molecules were kept to unity (Kundrot & Richards, 1987). The final R-factor was 15.6 o/0for 31,148 reflections between 1.9 A and 6.0 A. The results of this refinement are summarized in Table 1. In Fig. 1, the crystallographic R-factor of the 1.9 A structure is plotted as a function of resolution. Also plotted are theoretical curves corresponding to a mean co-ordinate error of 0.1, 0.2 and 0.3 A (Luzzati, 1952). The mean co-ordinate error derived from this plot is approximately 0.2 A. At high resolution. thr R-factor is greater than might be expected from the Luzzati curves. This is probably due to increasing measurement errors at this resolution. (d) Further

of the 23 ,ip strwturr enzyme-product complex

refinement

of

the

A structure of the enzyme-product, complex has been described by Schreuder et al. (1988a). This structure was now. in its turn. further improved on the basis of the refined 1.9 A structure of the PHBH F‘4D. pOHB First. 1.9 A st)ructurr of the complex. the PHBH . FAD. pOHB complex and the existing 2.3 A structure of the PHBH . FAD. 3.4.diOHB complex (Schreuder et al., 1988a) were compared. The differences between the models appeared to be localized in the regions that had been corrected during the refinement of the 1.9 A structure of the PHBH . FAD. pOHB complex. Therefore. a starting model for further refinement of the PHBH . FAD .3,4-diOHB complex was constructed as follows: the protein atoms were taken from the superimposed 1.9 A structure of the PHBH FAD. pOHB complex whereas the coenzyme FAD, the product 3,4-diOHB and the solvent molecules were taken from the existing structure of the PHBH . FSD .3,4-diOHB complex. Solvent molecules that were t’oo close to protein atoms in the new model were removed. After 19 cycles of Hendrickson-Konnert refinement and 2 rounds of model inspection and examination of solvent molecules, a final R-factor of 16.7y0 was obtained. A tot)al of 288 solvent’

H. A. Schreuder et al.

682

” 7”

I

0.35-

/

10.30

0.3010.20

b 0.25-

-0.10

Figure 1. R-factor (thick line), expressed as l/D where D is the Bragg spacing, as a function of resolution after curves for a refinement of the enzyme substrate (PHBH . FAD. pOHB) complex at 1.9 A resolution. The theoretical mean co-ordinate error of @l A, 0.2 A and 93 A are plotted in thin lines (Luzzati, 1952). molecules is included in the new model. The results are summarized in Table 1. Both the R-factor and the geometry of the new model are superior to those of the model reported by Schreuder el al. (19&z).

3. Results and Discussion (a) Improvements in the model of the enzyme-substrate complex During the model-building sessions in the highresolution electron density maps, it became clear that several loops in the 2.5 A model needed alterations. shows extensive Figure 2 the corrections of the main-chain atoms averaged per residue. Differences of about 3.5 A indicate loops that needed to be shifted by one residue. The residues involved are also depicted in Figure 3. It

appears that they are all near to each other in the “lower” part of the substrate-binding domain and are neither involved in FAD binding nor located in the active site region. The most important change made during the 1.9 A refinement in the active site region is the rotation of the peptide plane between Lys297 and Gly298 by 180”. This will be discussed further in the section about the active site. (b) Comparison of enzyme-substrate and enzyme-product complexes structural Since changes between the PHBH . FAD. pOHB and complex the PHBH . FAD. 3,4-diOHB complex may reveal interesting aspects of the catalytic mechanism of p-hydroxybenzoate hydroxylase, both complexes

Table 1 ReJinement statistics of the 1.9 d structure of the PHBH . FAD ’ pOHB complex and of the PHBH . FAD * 3,4-diOHB complex at 2.3 A resolution Enzyme-

Rt Reflections used Distance Bond distance Angle distance Planar 14 distance Planes Chiral volumes Non-bonded contacts Single torsion contacts Multiple torsion contacts Possible hydrogen bonds Thermal parameter correlations Main-chain bond Main-chain angle Side-chain bond Side-chain angle

substrate complex

Enzymeproduct complex

15.6 31.148

16.7 14.339

0.018 0.039 0.047 0.017 0.185

0.010 0.024 0.026 04Kl8 0.140

0.020 0.030 0.040 0.020 0.150

0.189 0.222 0.241

0.187 0.234 0.230

0.350 0.359 0.350

3.725 4.514 7.752 IO.535

3.328 4.516 7.244 10603

5.000 5400 7.006 10600

Target fs

These values are r.m.s. differences from ideal values (in A for distances, in AZ for thermal parameter correlations and in 6s for chiral volumes).

tR=

Wol - I~CII Wol

x 100%.

1.9 A Structure of p-Hydroxybenzoate

683

Hydroxylase

i-G 2.5 g 5

20 I.5 I.0 0.5

Residue

number

Figure 2. Positional corrections for the main-chain atoms, averaged per residue, between the 2.5 A model and the 1.9 A model of p-hydroxybenzoate hydroxylase. Loops with a shift of about 3.5 A had to be moved by 1 residue in the electron density in the course of the refinement process.

were compared by superimposing them. This superposition was performed using the method of Kabsch (1976), followed by removal of pairs of atoms deviating by more than three standard deviations, as in the method of Rao & Rossmann (1973). The models appeared to be related by a rotation of 0.8” and a translation of 1.0 A. In the of the PHBH . FAD. 3,4-diOHB new model complex (Schreuder et al., 1988a) no significant differences from the PHBH * FAD * pOHB complex were found. Even in the active site region (Fig. 11(b)), where in the one complex it is the substrate that is bound and in the other complex it is the product, the differences are small. The r.m.s. difference between C” co-ordinates after superposition of the new models of the PHBH . FAD. pOHB complex and the PHBH * FAD * 3,4-diOHB complex is only 0.2 A. However, some small differences are observed in the active site region, which will be discussed below. Because the model of PHBH * FAD * 3,4-diOHB complex was refined by using the 1.9 A structure of the PHBH . FAD. pOHB complex as a starting model, differences between both complexes could be masked. However, several observations indicate that this is unlikely. First, the refinement of the PHBH * FAD * 3,4-diOHB complex converged rapidly to a low R-factor (16.7%). Second, we did

not find residues in weak electron density, nor was there unoccupied density indicating a new position for a side-chain. Third, in studies on several thermitaseeglin-c complexes in our laboratory, which were carried out at a resolution and R-factors similar to those in the present paper, small differences between structures showed up nicely in electron density maps (P. Gros et al., unpublished results). The great similarity of the oxidized and the reduced PHBH . FAD . pOHB complex (Schreuder the et al., unpublished results) and PHBH . FAD. 3,4-diOHB complex may explain the fact that crystals of the PHBH . FAD. pOHB complex can be reduced and reoxidized to form product without loss of crystalline order (Van der Laan, et al., 1989; Schreuder et al., 1988a). (c) Main-chain

dihedral angles

The 1.9 A resolution data allowed the calculation of electron density maps that clearly indicated the repositioning of several carbonyl oxygen atoms. Eventually, all but three residues in the final refined model have phi-psi angle combinations within the allowed regions defined by Ramachandran & Sasisekharan (1968) (Fig. 4). The position of the residues with deviating phi-psi

Figure 3. Stereo drawing of the C” backbone of the 1.9 A structure of the PHBH . FAD. pOHB complex. Crosses denote residues with deviating phi-psi angles. Residues shifted by more than 3.0 A during the 1.9 A refinement are indicated by dots.

684

H. A. Schreuder

60.

. 0

et al.

.* ..

-

-60.

. .

-Is0

-120

c

J -60

0

60

120

Is0

Figure 4. Plot of the main-chain torsion angles phi and psi of the PHBH .FAD. pOHB complex. Regions allowed according to Ramachandran & Sasisekharan (1968) are indicated by broken lines. Open circles denote glycine residues, filled circles are non-glycine residues. The 13 non-glycine residues with phi-psi angles outside the allowed region are: Arg44, phi = 47”, psi = - 127”; AlaSO, phi = lo“, psi = 91”; and Asp144, phi = 86”, psi = - 102”. Ala80 and Asp144 are in turns having poor electron density, but Arg44 is well defined in the electron density map (see Fig. 5). angles in the three-dimensional structure are shown in Figure 3. Two of the three deviating residues, Ala80 and Asp144, have only weak electron density and the model may be less accurate at these positions. However, the density for the third amino acid residue with deviating phi-psi angles, Arg44, is well defined. At cycle 41 of the refinement procedure, this point was investigated in some detail. First, Arg44 and the adjacent peptides were removed from the model. Second, the resultant incomplete model was subjected to five cycles of least-squares refinement. The resultant electron density in a oA weighted 2F, - F, omit map clearly showed both peptide units and the Arg44 side-chain in essentially the same position as before this refinement, The final electron density and atomic model are shown in Figure 5. The peptide unit between Ile43 and Arg44 is rotated 180” with respect to the structure reported previously, making the phi-psi angle values even worse than in the earlier model (Schreuder et al., 198%). Interestingly,

this phi-psi

angle combination

is close to an

allowed region defined by Pullman et al. (1970), calculated with quantum chemical methods. Since the electron density is well defined (Fig. 5), we believe that the deviating phi-psi angles of Arg44 are real and not an artefact. (d) Secondary structure Due to the refinement with high-resolution data, the number of hydrogen bonds found in the protein

increased. Using the program DSSP from Kabsch & Sander (1983), we found 266 hydrogen bonds of the type NH to 0 in the 2.5 A structure and 295 in the 1.9 a structure. Hydrogen bonds involving atoms from the FAD, the substrate or bound solvent atoms were not counted. Since the main-chain hydrogen-bonding pattern is an important criterion for assigning secondary structure elements, the increase in the number of hydrogen bonds is accompanied by an increase in length of some a-helices and B-sheets. In Table 2 the a-helices and b-sheets found in the 1.9 A structure are given. The result of the automatic assignment by the Kabsch & Sander program is compared with visual assignments. It should be noted that the a-helices and b-sheets assigned by the program of Kabsch & Sander are in some cases shorter than the a-helices and P-sheets found by visual interpretation. The different secondary structure elements are depicted in Figure 6. For clarity, the three domains are drawn separately. As was pointed out by Richardson (1985), domains contiguous in space are not contiguous in sequence and vice veraa for p-hydroxybenzoate hydroxylase, making a choice between the two possible ways of domain assignment difficult. We have selected for domains that are contiguous in sequence. As a consequence, the domains do not have a compact globular shape, as they would have if we had selected for spatial contiguity. An interesting result of the increased number of

1.9 d Structure of p-Hydroxybenzoate

685

Hydroxylase

Figure 5. o,-weighted 2F,- F, (Read, 1986) stereo map contoured at the la level, together with the atomic model of Arg44, which has strongly deviating phi-psi angles. F, was calculated with the final enzyme-substrate co-ordinates.

hydrogen bonds found in the refined structure is that strand Dl and strand B3 fuse together to form one long P-strand and the same is true for strands B7 and D2. These two very long antiparallel strands (Fig. 6(b)) are sharply bent in the middle because residues 182 and 183 from the Dl/B3 strand bulge out and do not participate in the P-sheet hydrogen-bonding pattern. Instead they

form an outside bend, while the residues from strand B7/D2 continue their P-sheet hydrogenbonding pattern, forming the inside bend. (e) Binding

of FAD

Since this is only the second high-resolution structure of an FAD-containing protein (the 1st

Table 2 a-He&ices and b-sheets in p-hydroxybenzoate hydroxylase a-Helix Hl H2 H3 H4 H5 H6 H7 H8 H9 HlO Hll H12 H13

DSSP 12-24 36-40 5&58 63-68 88-91 102-l 16 164-167 23tC246 24%254 29&318 32&350 358-37 1 375386

Visual 1 l-26 35-42 4a61t 6249 87-94 lOlL117 164-169$ 235-246 248-255 297-319 32&351 357-374 374-387

/?-Strand

DSSP

Visual

Al A2 A3 A4 A5 A6 Bl B2 B3/Dl B4 B5 B6 B7/D2 Cl C2 c3 c4 D3 El E2 E3

5-8 2s-31 119-121 154-157 277-278 281-283 74-79 82-87 175-190 200-203 204-215 21&225 260-274 2 125130 138-143 14G151 28%290 4749 70-72 97-99

4-9 27-32 114-124 154-157 276279 28&284 73-80 El-88 174-191 198-203 206-216 217-224 26@275 l-5 125-131 137-143 14Gj-152 288-292 45-49 7%73 9G-100

The assignments of a visual inspection are compared with the assignments computer program DSSP, written by Kabsch & Sander (1983). t First and last turn somewhat wider than in a regular a-helix. j, Irregular helix.

obtained

by the

686

H. A. Schreuder et al.

4

FAD (c) Fig. 6.

1.9 A Structure of p-Hydroxybenzoate

Hydroxylase

687

Figure 7. Binding of the FAD molecule to p-hydroxybenzoate hydroxylese in stereo. The protein bonds are indicated by open bars, the FAD and p-hydroxybenzoate molecule are indicated by filled bars. Concentric circles denote bound solvent molecules and potential hydrogen bonding interactions are denoted by broken lines. The adenine pocket is filled with a number of solvent molecules and most of the polar interactions between the adenine and the protein are via these water molecules. The puckering of the ribose is C2’ endo. The Figure is drawn with co-ordinates from the enzymesubstrate complex. The enzyme-product complex is virtually identical, except for a rotation of the product, 3,4-diOHB, replacing the substrate, pOHB.

being glutathione reductase (Karplus & Schulz, 1987)), it is of interest to describe the FAD binding in some detail. The FAD is bound in an extended conformation, spanning approximately half the length of the protein. The ADP moiety of the FAD is bound by a flab-fold formed by the first 32 residues of the enzyme (Fig. 6(a)). This type of fold is found in many nucleotide-binding proteins (Rossmann et al.,

1975; Wierenga et al., 1985). In addition to interactions between enzyme and numerous coenzyme, as many as nine solvent molecules are involved in hydrogen bonding with the FAD molecule. With one exception, all these solvent molecules are involved in three or more potential hydrogen bonds. A detailed picture of the FADbinding site is given in Figure 7. A total of 31 possible hydrogen bond interactions of the FAD

Figure 6. Drawing of the secondary structure elements and overall folding of the 3 domains of the p-hydroxybenzoate hydroxylase molecule. The 3 domains are slightly rotated with respect to each other to allow an optimal view. (a) Domain 1: the FAD-binding domain. The chain starts at the left of the Figure with strand Cl and ends at residue 158 at the end of strand A4. The piece of polypeptide chain from residue 158 to Lys175, including helix H7, also belongs to the 1st domain, but for clarity it has been drawn in the 2nd domain. The Al-Hl-A2 flaj?-unit in the left part of this domain is similar to nucleotide-binding units in a number of other proteins (Wierenga et al., 1985). (b) Domain 2: the substrate-binding domain. This domain starts after residue 175 at the beginning of strand Dl and ends at residue 290 at the end of strand D3 in the centre of the picture. The piece of polypeptide chain from 158 to 175 belongs to domain 1. Virtually all residues that interact with the substrate are located in domain 2. Of the substrate-binding residues mentioned in Table 7, Tyr201 is part of strand B4; Ser212 and Arg214 are part of strand B5 and Tyr222 belongs to strand B6. All these residues protrude at an approximately perpendicular angle from sheet B. Pro293 only is not located in domain 2, but in the active site loop between domain 2 and domain 3. (c) Domain 3: the interface domain. All contacts between the subunits of the p-hydroxybenzoate hydroxylase dimer are mediated via this domain. Helix Hl 1 is in contact with helix H12 of the 2-fold-related molecule. Domain 3 starts after residue 290 in the centre of the Figure. The chain immediately forms the active site loop, which is sharply bent, possibly due to 2 consecutive proline residues at positions 292 and 293. The chain ends at residue 391 in the top centre of the Figure. The last 3 residues (392 to 394) are not visible in the electron density map, probably due to disorder. The most prominent features in this domain are the very long helices HlO and Hll, each comprising about 20 residues (Table 2). The polypeptide chain between HlO and Hl 1 is drawn as a C” tracing to illustrate a piece of 3,c helix preceding a-helix Hl 1.

H. A. Schreuder et al.

688

Potential

Table 3 hydrogen-bonding interactions between FAD and hydroxylase and solvent molecules

Atom 1

Atom 2

A. Flawin ring N-l (3.2) N-l (3.1) N-l (3.3) o-2 (3.0) o-2 (3.1) o-2 (3.1) N-3 (2.9) o-4 (3.1) o-4 (3.1)

Sol717 N N N N-D2 N 0 N N

B. Ribityl

Further

p-hydroxybenzoate

hydrogen bonds made by the solvent molecule listed in the column under atom 2

Sol579 (2.4), N Gly298 (2+J), N Lys297 (3.3) La299 Gly298 Leu299 Asn300 Asn300 Va147 Gly46 Va147

chain

o-2 (2.6) O-El O-3’ (2.8) O-D1 O-3’ (3.2) N O-4’ (2.7) N-H2 O-4’ (2.9) N-E2 C. Ribityl 5’-phosphate O-F1 (2.5) so1593 O-F1 (3.1) N O-F2 (2.9) so1419 O-F2 (3.1) N D. AMIV ribosc 5’-phosphate O-Al (2.5) Sol615 O-Al (3.3) N-H2 O-A2 (2.7) Sol402 O-A2 (2.8) Sol596 E. AMLN ribose O-3’A (2.4) O-El (2.7) N-H2 0-3’A O-3’A (3.2) O-E2 0-2’A (2.6) O-E2 0-2’A (2.7) N-H2 F. Adeninr N-6A (2.9) Sol607 X--6A (3.2) Sol404 S-1A (3.2) Sol616 (3.2) N-H2 s-34

Gln102 Asp286 Leu299 Arg44 Gln102 0 Ala284 (2.8), N Gly160 (3.0), 0 Cys158 (3.3) Asp286 0 Cys158 (2.5), N Glyll

(2.9), N Gly14 (3.0)

SW1 3 NH, Arg44 (2.4), Sol612 (2.7). N-E Arg42 (2.7) Arg44 Sol602 (2.8), So1690 (3.2) O-D2 Asp286 (2.9), N Phel61 (3.0) GM32 Arg42 Glu32 Glu32 Arg33 0 Va1127 (2.9), Sol403 (3.0) 0 His162 (3.2) Sol403 (2.8). 0 Ala124 (3.1), N Val127 (3.1) Arg33

The hydrogen-bonding interactions of these solvent molecules involved with t,he protein are given as well. Distances in A are given in parentheses. The nomenclature of the FAD atoms is given in Fig. 8.

with protein and solvent atoms is listed in Table 3, while van der Waals’ interactions are given in Table 4. Details of the binding of the different parts of the FAD molecule will be discussed in the next paragraphs. (i) The adenine-ribose moiety The adenine binding pocket in p-hydroxybenzoate hydroxylase does not seem to be very specific. because most polar interactions are mediated by solvent molecules. The adenine ring makes three hydrogen bonds with solvent molecules and only one with a protein atom. The latter involves the side-chain of Arg33 and the N-3 atom of t,he adenine (Table 3). The adenine NH, group is hydrogen bonded to Sol607 and to Sol404. These two solvent molecules interact’ further with mainchain carbonyl oxygen atoms. The N-l of the adenine interacts with So1616, which is making hydrogen bonds with two protein atoms (Table 3). The enzyme appears to employ an “indirect”.

water-mediated mode of binding for the adeninr ring. The ribose moiety is tightly fixed by t’he protein. The 2’ and 3’ hydroxyl groups of the ribose are sandwiched between the guanidine group of Arg33 and the carboxylate group of Glu32. At least four strong hydrogen bonds are present between the two ribose hydroxyl groups and the charged side-chains of Arg33 and Glu32 (Table 3). The O-4’ oxygen of the ribose (0 in Fig. 8) is not involved in hydrogen bonds. (ii) The pyrophosphate group The pyrophosphate group bears two formal negative charges that, must be “solvated” by the protein. The negative charge is in party compensated by the dipole of helix HI in the j?c@fold at the N terminus of p-hydroxybenzoa,te hydroxylase (Fig. 6(a); Wierenga et al., 1985). Further charge compensation is realized by two positively charged side-chains. Of these, only Arg44 makes a direct, salt

1.9 B Structure of p-Hydroxybenzoate

C3-C2

01

l

Hydroxylase

hydrogen bonds to the buried solvent molecule Sol593 and to the main-chain nitrogen atom of Asp286. Sol593 is within hydrogen-bonding distance of three other ligands: the carbonyl oxygen atom of Ala284, the peptide nitrogen atom of Gly160 and the carbonyl oxygen atom of Cys158. These four possible hydrogen bonds can be made only if the solvent is a protonated water molecule. The distance of the solvent molecule to the carbonyl oxygen atom of Cys158 is rather long (3.3 A), however, and it is possible that only three hydrogen bonds are actually being made. The ribityl phosphate oxygen atom, O-A2. is hydrogen bonded to the solvent molecules Sol402 and So1596. Since the pyrophosphate group makes five potential hydrogen bonds with solvent molecules and three with protein atoms, it can be concluded that solvent molecules are important for binding of the pyrophosphate group. One might say that the enzyme does not bind a pyrophosphate moiety as such, but a rather solvated pyrophosphate group. (iii) The ribojavin

Figure 8. Nomenclature of the atoms of the FAD, the pOHB and the 3,4-diOHB molecule. The torsion angles of the FAD molecule are indicated as well. The Figure and nomenclature of the FAD are taken, with permission, from Schulz rt a.Z.(1982).

bridge to the O-Al of the pyrophosphate group (for nomenclature, see Fig. 8). The guanidine group of the second arginine, Arg42, interacts indirectly via Sol615 with the pyrophosphate moiety. p-Hydroxybenzoate hydroxylase contains the sequence Gly-X-Gly-X-X-Gly, where X denotes a non-glycine residue. A database search has revealed that this pattern, or fingerprint, of glycine residues is conserved in many proteins containing a flc$ nucleotide binding fold (Wierenga et al., 1986). In our protein, this pattern occurs at the N terminus of helix Hl. O-F2 of the pyrophosphate group is hydrogen bonded directly to the peptide nitrogen atom of Serl3, which is in the fifth position of the Gly-X-Gly-X-X-Gly sequence. The phosphate oxygen atom O-F2 is also hydrogen bonded to So1419. This solvent molecule interacts further with the carbonyl oxygen atom of Cys158 and with the peptide nitrogen atoms of two conserved glycine residues, Glyll and Gly14 at positions 3 and 6 of the fingerprmt sequence. Therefore, the two conserved glycine residues mentioned do not interact directly with the pyrophosphate group, but via a bound solvent molecule. However, the pyrophosphate is situat#ed near the N terminus of the “phosphate binding” helix Hl, its charge interacting favourably with the helix dipole (Ho1 et al., 1978: Hol, 1985). Other interactions of the pyrophosphate group involve mainly solvent molecules. O-F1 makes

689

moiety

In contrast to the adenosine moiet’y, only one solvent molecule is hydrogen bonded to the riboflavin moiety. This is So1717, located at 3.2 A from the N-l of the flavin ring. The ribityl chain is bound by the side-chains of Arg44. Gln102 and Asp286, and by the main-chain nitrogen of Leu299. The flavin ring is within hydrogen-bonding distance of five main-chain nitrogen atoms, one main-chain oxygen atom and the side-chain amide nitrogen atom of Asn300, so the interactions of the flavin ring are almost exclusively with main-chain atoms of the protein (Tables 3 and 4). Six out of eight peptides within 3.5 A of the flavin ring are oriented with their nitrogen atom directed towards the ring. Three of them (containing the main-chain nitrogen atoms of Lys297. Gly298 and Leu299) are part of the macro-dipole of helix HlO (Fig. 6(c)). The flavin is thus surrounded by a number of partial positive charges that originate from polarized peptide units. We expect that the resultant electrostatic effect will provide stabilization of negatively charged flavin intermediates. This is quite interesting because it is known from nuclear magnetic resonance studies t,hat the reduced flavin in p-hydroxybenzoate hydroxylase bears a negative charge at’ the N-l position (Vervoort et aZ., 1986). (iv) Torsion angles of the fi’A I> The FAD molecule contains 13 single bonds about which rotations are allowed. The torsion angles about, these bonds are indicated in Figure 8, while their values are listed in Table 5. For comparison, the torsion angles of t’he FAD in the 1.54 A structure of glutathione reductase are given as well (Karplus & Schulz, 1987). The torsion angles of the coenzyme in both structures are strikingly similar. the average difference being 8”. The largest’ difference is 25” for +i\. This value is significantly smaller than the differences. up to 88”, found

690

H. A. Schreuder et al.

Table 4 van der Waals’ interactions between p-hydroxybenzoate and FAD Component of FAD

Contact in p-hydroxybenzoate

Flavin ring

Ribityl

Ala296Leu299 Ala45 p-Hydroxybenzoate Arg44 Gln102 Asp286 Gly298 Prol2-Ser13 Glu32 Arg42 Gly9 Asp150 Asp159 Gly163 Be164 Arg33

chain

Ribityl 5’.phosphate AMN ribose

Adenine

van der Waals’ interactions

(f) Planarity

of the jlavin

Main-chain CA, CB C-6 Side-chain O-E2 Side-chain Main-chain Side-chain Side-chain Side-chain CA CA 0

;pG, N, CB

defined by interatomic

between the 2.0 A structure and the 1.54 A structure of glutathione reductase (Schulz et al., 1982; Karplus t Schulz, 1987). The similarity in the conformation of the FAD, observed between the 2.5 A structure of p-hydroxybenzoate hydroxylase, and the 2-O A structure of glutathione reductase (Wierenga et al., 1983), has become even greater after refinement of both structures at higher resolution. This great similarity is remarkable, since the three-dimensional structures of the domains interacting with the ribityl chain and the flavin ring have no similarity whatsoever in the two proteins. ring

In the structures of p-hydroxybenzoate hydroxylase reported by Wierenga et al. (1979) and Schreuder et al. (1988a), all atoms of the flavin ring were restrained during refinement to lie in a single plane. During the present refinement, however, it was observed that the flavin ring did not become completely planar, despite imposed planarity restraints. In particular, the O-4 atom (Fig. 8) was lying somewhat out of the plane. It appeared from the electron density that the flavin was not fully planar but was slightly twisted. Therefore a different scheme of restraints was introduced during refinement of the present structures. The benzene ring and the pyrimidine ring were restrained to be planar, but no planarity restraints were imposed on the central ring of the flavin moiety. With these restraints, the benzene ring and the pyrimidine ring are no longer necessarily coplanar. Refinement using these new restraints resulted in an angle of 10” between the benzene and the pyrimidine rings for the 1.9 A structure of the PHBH * FAD * pOHB complex and an angle of 19” between the two planes structure in 2.3 A of the the PHBH . FAD. 3,4-diOHB complex. This angle is largely due to a propeller type of twisting of the

hydroxylase

distances of less than 3.5 A.

Aavin ring and not to a butterfly type of bending, as was observed in trimethylamine dehydrogenase (Mathews & Lim, 1987). The much larger twist of the flavin ring observed in the PHBH * FAD. 3,4-diOHB complex in comparison with the twist of the flavin in the PHBH . FAD * pOHB complex is mainly due to an extra rotation of N 13” of the benzene ring in the PHBH . FAD. 3,4-diOHB complex. The temperature factors of the flavin ’ t,he PHBH . FAD *3,4-diOHB complex are r:her high, however, and the intraplanar angle obtained is therefore not very accurate. In Figure 9, a stereo picture of the electron density and atomic model of the flavin ring is given for both structures. Intuitively, one would expect the oxidized flavin ring to be fully planar, but this study and a number of other X-ray diffraction studies as well as some

Table 5 Torsion angles of FAD in p-hydroxybenzoate hydroxylase reductase and in glutathione reductase (Karplus & Schulz, 1987)

Angle?

p-Hydroxybenzoate hydroxylase (deg. 1

Glutathione reductase (deg.)

134 -57 162

59 87 55 72 -154

179 161 -171 -170

-88 t A definition

of the angles is given in Fig. 8

139 - 73 163 59

112 41 75

-150 179 176 --176 -175 -76

1.9 A Structure of p-Hydroxybenzoate

Hydroxylase

691

(a)

(b) Figure 9. Electron densities and atomic molecules of the flavin ring in p-hydroxybenzoate hydroxylase in stereo: (a) in the 2.3 A structure of the PHBH .FAD. 3,4-diOHB complex and (b) in the 1.9 A structure of the PHBH . FAD. pOHB complex. The view is along the plane of the flavin ring. The a,-weighted 2F,,- F,, maps (Read, 1986) contoured at the la level are also shown

nuclear magnetic resonance investigations, report oxidized flavin rings that are not completely planar. A few examples are the following. The angles between the benzene and the pyrimidine ring in model compounds for oxidized flavin are 18” for 3-methyllumiflavin and 7-O” for 9-bromo-3,7,8,10-tetramethylisoalloxazine (Norrestam & Stensland, 1972; Von Glehn & Norrestam, 1972). An angle between both planes of 3.3” is found in the 1.54 A structure of glutathione reductase (Karplus & Schulz, 1987). In the 1.8 A structure of Clostridium MP flavodoxin a bending angle of 4.2 to 8.6” is observed (Laudenbach et al., 1987). Nuclear magnetic resonance

studies

by Miiller

and co-workers

Moonen et al., 1984; Vervoort et al., 1986; Vervoort, 1986). Vervoort et al. (1986) and Vervoort (1986), however, observe a decreased sp2 character of the N-10 of the flavin ring as it is bound to bacterial luciferase. They infer that the N-10 has moved out of plane. Such a movement may cause distortions of the coplanarity of the flavin ring system. The largest deviation from planarity is a bending angle of 20” reported for the 2,5 A structure of trimethylamine dehydrogenase (Mathews & Lim, 1987). The value of the angle is comparable to the value obtained in the PHBH. FAD. 3,4diOHB complex. However, as stated before, the flavin ring in p-hydroxybenzoate hydroxylase is twisted like a propeller, while the flavin ring in trimethylamine dehydrogenase is bent like a

on a

number of flavoproteins indicated a more or less planar flavin ring (Van Schagen & Miiller, 1981;

Table 6 Comparison of the lengths of the hydrogen bonds between the O-4 of the jiavin ring and protein nitrogen atoms in various models of p-hydroxybenzoate hydroxylase Length of hydrogen bonds (A)

Peptide nitrogen of Gly46 Va147

Enzyme-product

complex

Planar flavint

Non-planar flavinl

3.48 3.36

3.12 3.10

Enzymesubstrate complex non-planar flavinj 3.05 3.11

t Schreuder et al. (1988a). 1 This work. 3 Schreuder et al. (198%). 11The model compound was 4,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin 1978)

Flavin 4a-hydroperoxide model compounds§ll 2.76 2.95

(Bolognesi

et al..

H. A. Schreuder et al. butterfly. The observation of a number of nonplanar flavins shows that no excessive energy is required for the flavin ring to become non-planar. Quantum chemical calculations gave an energy of 2.9 kcal/mol (1 cal=4.184 J) for a lo” bending and an energy of 85 kcal/mol for a 20” bending (Dixon et al., 1979). Interestingly, the deviations from planarity of the flavin ring in p-hydroxybenzoate hydroxylase are in the same direction as for the flavin 4a-hydroperoxide model compound reported by Bolognesi et al. (1978). In other words, the flavin has adopted a conformation more similar to that of the flavin 4a-hydroperoxide intermediate than a completely planar flavin ring. This probably results from the fact that the active site of p-hydroxybenzoate hydroxylase is complementary to the Gavin 4a-hydroperoxide intermediate as it occurs during the catalytic reaction of the enzyme (Schreuder et al., 1988b). The largest difference observed in our models between planar and non-planar flavins in p-hydroxybenzoate hydroxylase occurs around the O-4 atom of the flavin. The lengths of possible hydrogen bonds between the O-4 of the flavin and main chain nitrogen atoms of Gly46 and Va147 are given in Table 6. Ths shortest and probably strongest hydrogen bonds are made with the flavin 4a-hydroperoxide model compound. The energy necessary for bending of the flavin ring (Dixon et al., 1979) is comparable to the energy gain that can be expected from stronger hydrogen bonds at O-4 (Schreuder et al., 19883). (g) Active site geometry Although the overall conformation of the active site is very similar to what has been reported (Schreuder et al., 1988a), some small but probably significant corrections in the structure were incorporated during refinement. Figure 10 is a

picture of the electron density and atomic model of the active site region of p-hydroxybenzoate hydroxylase. The electron density is generally of good quality. An important correction in the protein structure was the flipping over of the peptide group between Lys297 and Gly298. Instead of the carbonyl oxygen atom of Lys197, the nitrogen of Gly298 is now pointed towards the flavin ring. An extra solvent molecule could be placed near the position previously occupied by the carbonyl oxygen of the peptide unit. The result is that two solvent molecules (Sol579 and So1717; Fig. 11) are bound in the entrance to the proposed nicotinamide-binding pocket next, t,o the flavin ring. All peptide groups of the active site loop, comprising residues 295 to 300, are oriented with their nitrogen atoms directed towards either the flavin ring or the active site cavity. The nitrogen atoms of Gly298 and Leu299 are within hydrogenbonding distance of the flavin N-l. The peptide nitrogen atoms of Lys297 and Gly298 are pointed towards So1717 and have a favourahle orientation for the formation of hydrogen bonds (Fig. 11). The peptide nitrogen atoms of Gly295 and Ala296 are pointed towards the active site cavity and are involved in the binding of So1562, which is also within hydrogen-bonding distance of the O-G of Ser342 and the carbonyl oxygen of Pro292. This hydrogen-bonding network is illustrated in Figure 11(a). Visser (1983) proposed on the basis of t,he partially refined 2.5 A structure that the peptide nitrogen atoms of Ala196 and Lys297 might function as an “oxyanion hole” that, binds the peroxide anion during catalysis. In the present model, Sol562 is bound in this site, close to three peptide nitrogen atoms, but also close to the carbonyl oxygen atom of Pro292 and the 0-G of Ser342 (Fig. 11(a)). From the present structure. it, seems more likely that the peroxide anion is located near the position of So1717, because this position is

Figure 10. Electron density, in stereo, in the active site of the PHBH . FAD .pOHB complex. The a,-weighted map was contoured at the la level. In the centre the flavin ring is visible with the substrate molecule below.

2F,-F,

The crosses indicate

the position

of 2 bound

solvent

molecules.

1.9 A Structure of p-Hydroxybenmate

Hydroxylase

693

(b) Figure 11. The active site of p-hydroxybenzoate hydroxylase in stereo. (a) Hydrogen-bonding network between the N-l of the fiavin ring, 3 bound solvent molecuies and several protein atoms. The protein is indicated by open bonds, the flavin by filled bonds. Concentric circles indicate bound solvent molecules and broken lines denote potential hydrogen bonds. Also visible at the bottom is a hydrogen bond between the peptide nitrogen atom of Ala296 and the carbonyl oxygen atom of Pro293, which defines a sharp turn in the active site loop. (b) Superposition of the active site of the lines indicate the PHBH . FAD . pOHB complex on to the PHBH . FAD .3,4-diOHB complex. Continuous PHBH . FAD .pOHB complex; broken lines indicate the PHBH . FAD. 3,4-diOHB complex. x , position of solvent molecule in PHBH . FAD. pOHB complex; + , position of solvent molecule in PHBH . FAD. 3,4-diOHB complex.

closer to the flavin ring and to the positive end of the dipole of helix HlO. In the 2.5 A PHBH . FAD. pOHB structure, the plane of the substrate was rotated 21” approximately around the C~,&,) axis with respect to the product ’ plane of the the PHBH . FAD * 3,4-diOHB complex (Schritder et al., 1988a). After refinement against 1.9 b resolution data, this rotation is reduced to 14”, which can be examined in Figure 11 (b), where a superposition of the active sites of the PHBH . FAD. pOHB and the PHBH . FAD. 3,4-diOHB complexes is given. The number of hydrogen bonds between the substrate or product molecule and the protein (Table 7) has increased after refinement. The side-

chain of Arg214 has been rearranged slightly so that both NH2 groups are now able to make hydrogen bonds with both carboxyl oxygen atoms of the substrate or product molecule. The mainchain atoms of Pro293 and Thr294 have moved a little, which brings the carbonyl oxygen atoms of Pro293 and Thr294 within hydrogen-bonding distance of the 4-OH of the substrate or product molecule. Since the carbonyl oxygen atom of Pro293 seems to be better positioned, a hydrogen bond between the 4-OH of the substrate and this carbonyl oxygen is probably present. These newly discovered hydrogen bonds increase the number of hydrogen bonds between the substrate and the protein from four to six and between the product

694

H. A. Schreuder et al

Table 7 Potential hydrogen bonds of p-hydroxybenzoate (substrate) and 3,4-dihydroxybenzoate (product) with p-hydroxybenzoate hydroxylase Substrate or product atom

Distance (A) to Atom/ Protein

&F”P

Tyr201 Pro293 Thr294 Pro293 Ser212 Arg214 Tyr222 Arg214

OH

o-4 o-4 o-4 o-3

0 0 0

o-1* o-1* o-2* 0-2*

O-G NH, OH NH,

The nomenclature

Substrate

Product

2.57

2.54 3.24 3.30 2.35 2.95 3.01 2.36 3.07

2.92 2.97 2.75 2.83 2.33 3.07

used for the atoms is given in Fig. 8.

and the protein from five to seven. The short hydrogen bond of 2.35 A between the 3-OH of the product and the carbonyl oxygen of Pro293 is still present after refinement. Also, the short distance between the OH group of Tyr222 and one of the carboxyl oxygen atoms of the substrate and product molecule (-2.35 A) remains present. The broad and continuous electron density between the OH of Tyr222 and this carboxyl oxygen atom suggests a strong hydrogen bond. Since this short contact occurs in both the and the PHBH . FAD +pOHB PHBH * FAD. 3,4-diOHB complex, it does not influence the relative binding constants of substrate and product.

PHBH . FAD .3,4-diOHB complex (Schreuder et al., 1988a). All regions having average main-chain B-factors greater than 30 A2 are exposed loops at the surface of the protein. Some loops having high B-factors are the following: (1) the loop containing Ala80, which has deviating phi-psi angles, is at a sharp t,urn between strands Bl and B2; (2) the loop around Asp144, which has also deviating phi-psi angles, connects strands C2 and C3 (Ala80 and Asp144 are indicated in Fig. 3); (3) the loop 227-234 is a fully exposed stretch of amino acid residues, having a 3,, helix hydrogen-bonding pattern; (4) the region around residues 355 to 356 is a short loop. connecting helix Hll and helix H12; (5) finally, the thermal factors increase sharply at residue 390. Although the terminal residues 392 to 394 were included in the previous 2.5 A model, their density became so weak during refinement that they were omitted from the final 1.9 A model. We conclude t,herefore that the three C-terminal residues are disordered. It is interesting to notice that,, in the enzyme--~ substrate complex, the active site loop 288-300 has amongst the lowest B-factors of the whole molecule. Also the substrate (average B= 15.7 A2) and the flavin ring (average B= 16.6 AZ) have relatively low B-factors. This means that the flavin ring, the substrate and some surrounding residues are tightly fixed by the rest of the protein. In the newly refined enzyme-product complex, the product 3,4-diOHB has low B-factors (average B = 12.8 A’), whereas the avera e B-factor of the flavin ring is rather high (31.8 A B). It is not easy t,o find an explanation for this observation.

(h) Thermal motion and disorder Inclusion of high-resolution data in the refinement led to significantly higher temperature factors of the 1.9 A model as compared to the 2.5 A model. The average B-factor increased from 12.3 dz for the 2.5 A structure to 24.7 A2 for the 1.9 i% model. The distribution of thermal motion over the polypeptide chain (Fig. 12) shows a pattern similar to the earlier for the reported distribution

Residue

Figure. 12. Isotropic the B-factors

(i) Possibility

of bound ions

The putative ions bound to the enzyme-product complex as mentioned by Schreuder et al. (1988~) were not substantiated during refinement against 1.9 w data. Some spherical blobs of density are still present at distances greater than 3.5 A from protein atoms or other solvent molecules, but neither the height nor the shape of these electron density

rumber

temperature factors (B) of the 1.9 A structure of the main-chain atoms, averaged per residue.

of the PHBH

. FAD.

pOHB

complex.

Plotted

are

1.9 A Structure of p-Hydroxybenzoate features seems to indicate a tightly bound sulphate, phosphate or other ion. Also none of the B-factors molecules became of the assigned solvent exceptionally low, which would otherwise have been an indication of a bound ion. These results indicate that no strongly bound ions are present, but obviously some of the solvent molecules included in the model may represent mobile and/or weakly occupied sulphate, phosphate or other ions. The competition between sulphate and phosphate ions for the NADPH-binding site (Wijnands et al., 1984) arises most likely from relatively weak or non-specific interactions of many of these ions with residues involved in NADPH binding and not from tight binding of such an ion to a specific site.

4. Conclusions Refinement of the structure of the p-hydroxybenzoate hydroxylase-substrate complex against 1.9 A data obtained with synchrotron radiation has led to a substantial improvement of the atomic model, as can be judged by for example the crystallographic R-factor and the Ramachandran plot. Incorporation of the improvements from this model into the model Of the PHBH. FAD. 3,4-diOHB complex has led to a better model of the latter as well. We expect the 1.9 A model to be an excellent starting point for the refinement of other complexes along the catalytic cycle of p-hydroxybenzoate hydroxylase. In addition, the improved definition of the active site, including two water molecules bound at van der Waals’ distance of the flavin ring, provides a new basis for discussions about the complex reaction mechanism of p-hydroxybenzoate hydroxylase. It is a pleasure to acknowledge the help of Drs F. M. D. Vellieux and H. Terry for assistance with the data collection at the Hamburg synchrotron, M. B. A. Swarte, H. Groendijk and H. Kelders for scanning the oscillation films and Drs J. M. Van der Laan, J. Vervoort and W. J. H. van Berkel for helpful discussions. We are indebted to Professor F. Miiller for the critical reading of this manuscript and helpful suggestions. We thank Carmen Rayner for the drawing in Fig. 6. Most of the computations were carried out on the Cyber 205 supercomputer in Amsterdam under a grant for computer time by the “Werkgroep Gebruik Supercomputers”. This research has been carried out under the auspices of the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).

References Bolognesi, M., Ghisla, S. & Incoccia, L. (1978). Acta Crystallogr. sect. B, 34, 821-828. Dagley, S. (1982). In Flawina and Flawoproteins (Massey, V. & Williams, C. H., eds), pp. 311-317, Elsevier Scientific Publishing Co., Amsterdam.

Hydroxylase

695

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