[24] Hydroxybenzoate hydroxylase

138

HYDROCARBONS

AND RELATED

[24] H y d r o x y b e n z o a t e

[24]

COMPOUNDS

Hydroxylase

By BARRIE ENTSCH There are three isomers of hydroxybenzoate, and each is hydroxylated by a separate enzyme in microorganisms. These enzymes are intraceUular flavoprotein monooxygenases which require reduced pyridine nucleotide for the reaction. They channel aromatic compounds into the metabolic pathways which open the benzene ring to form common intermediates of energy metabolism. The enzyme which hydroxylates 4-hydroxybenzoate (4-hydroxybenzoate 3-monooxygenase, EC 1.14.13.2; p-hydroxybenzoate hydroxylase) has been studied extensively over the past 20 years and is the subject of this chapter. The enzyme which hydroxylates 2-hydroxybenzoate [salicylate monooxygenase (hydroxylase), EC 1.14.13. l ] is increasingly the subject of investigation at present at the molecular level.l.2 Two enzymes hydroxylate 3-hydroxybenzoate (classified under EC 1.14.13.23 and 24). The enzyme which hydroxylates para to the substrate hydroxyl group is now under investigation? The biological reaction catalyzed by p-hydroxybenzoate hydroxylase is represented by the following equation: HO HO-~COO-

+ NADPH + n + + 02 --~ H O - ~ C O O -

+ NADP + + H20

A wide range of soil microorganisms seem to be capable of this reaction or the same reaction with NADH. However, it is the enzyme from the fluorescent pseudomonads which has been studied in detail. The enzyme is not normally produced except in the presence of its substrate as a source of carbon. A previous report on this enzyme from Pseudomonas fluorescens appeared in this series in 1978.4 Since that time, much information has appeared in the literature, and the enzyme has become even more established as a model for flavoprotein oxygenases. This chapter presents an improved method of preparation, a selected summary of properties established since the previous contribution in this series, and some information on the gene for the enzyme. 1H. Kamin, R. H. White-Stevens, and R. P. Presswood, this series, Vol. 53, p. 527. 2 G. H. Einarsdottir, M. T. Stankovich, and S.-C. Tu, Biochemistry27, 3277 (1988). 3 L.-H. Wang, R. Y. Hamzah, Y. Yu, and S.-C. Tu, Biochemistry26, 1099 (1987). 4 M. Husain, L. M. Schopfer, and V. Massey, this series, Vol. 53, p. 543.

METHODS IN ENZYMOLOGY, VOL. 188

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form r~rved.

[24]

HYDROXYBENZOATE HYDROXYLASE

139

Assay Method The assay mcthod has remained essentiallyunchanged in its present form since it was developed by Entsch et al.5 from a knowledge of the p H and buffcr ion depcndcncc of the enzyme. The assay is reliableeven with crude cellextracts. Principle. Activity is measured spcctrophotomctrically by following the rate of p-hydroxybenzoate-dcpcndcnt oxidation of N A D P H at 340 n m in air-saturatedbuffer at 25 ° Procedure. Assays arc conducted at 25 ° in l-cm ccUs in a recording spcctrophotomctcr. The reaction solution of 3 ml (or less)contains 33 m M Tris as sulfatc salt, 0.33 r n M E D T A as sodium salt, 0.33 m M sodium p-hydroxybenzoate, 0.23 m M N A D P H , 3.3 ~ FAD, and 0.26 m M oxygen, all at p H 7.9 to 8.0. The reaction is initiated by the addition of enzyme, and the N A D P H oxidation rate is measured. Alternatively, the rate of reduction of oxygen can bc measured in an oxygcn clcctrodc chamber. It is important to purify p-hydroxybcnzoic acid for the reaction by recrystallizationfrom hot water. F A D is necessary to kccp the cnzymc saturated with cofactor at the low enzyme concentrations in the assay. Units. One unit of enzyme is the amount that oxidizes I /imol of N A D P H or reduces I/tmol of oxygen per minute under the assay conditions. Specific activity is cxprcssed as units of cnzymc per milligram of protein. Purification The procedure presented below has been evolved from the methods of Entsch et aL 5 and Miiller et aL 6 and it is effective in the purification of enzyme from P. fluorescens and Pseudomonas aeruginosa rapidly in high yields. A recent report 7 on enzyme from Corynebacterium cyclohexanicum suggests that the affinity step below is not effective in the purification of enzyme from all sources. An alternative procedure is thus presented in that reference. Although enzyme has been studied in the past from P. fluorescens, the procedure below is written for enzyme from P. aeruginosa, since we have found that the enzyme from the latter bacterium is almost identical to that from P. fluorescens (see Genetics section) but is induced to higher concen-

5 B. Entsch, D. P. Ballou, and V. Massey, J. Biol. Chem. 251, 2550 (1976). 6 F. Miiller, G. Voordouw, W. J. H. van Berkel, P. J. Steennis, S. Visser, and P. J. van Rooijen, Fur. J. Biochem. 101, 235 (1979). 7 T. Fujii and T. Kaneda, Eur. J. Biochem. 147, 97 (1985).

140

HYDROCARBONS AND RELATED COMPOUNDS

[24]

trations in cells of P. aeruginosa. In addition, the biology of P. aeruginosa has been studied extensively because of its medical significance. Growth of Bacteria. Pseudomonas aeruginosa PAO 1C (ATCC 15692) is maintained for extended periods when 1 volume of culture in the medium below is added to 2 volumes of glycerol and stored at - 7 0 °. Cells are revived for cultures by streaking on plates containing the medium below plus 1.5% agar at 30 ° (allow 2 days). Colonies of cells are used to start liquid cultures as soon as the cells grow up. Plates are not stored. Cells can be grown in continuous culture or in batch culture. Only batch culture is described here. Any volume of culture can be grown, depending on the facilities for aeration. Growth rates are normally limited by oxygen supply. The growth medium contains the following in 1.0 liter: 3.5 g ofp-hydroxybenzoic acid, 4.0g of NH4NO3, 1.0g of NH4C1, 0.22g MgSO4" 7H20, and 3.5 g K2HPO4. The pH is adjusted to 7.0 with NaOH. Iron (0.5 mg) is added as the EDTA complex (1 mol of iron to 3 mol of EDTA), and micronutrients are as follows: 0.25 mg of CuSO4" 5H20, 0.35mg of ZnSO4"7H20, 0.35mg of MnC12-2H20, 2.0mg of CaC12" 2H20, 0.45 mg of Co(NO3)2" 6H20, and 0.25 mg of ammonium molybdate. The iron and micronutrient solutions are sterilized separately and added when the macronutrient solution is cool. Cultures are grown in two stages at 32 to 35 °. A starter culture is grown from plates, and this provides an inoculum of about 5% in the main culture. Cells are harvested before the substrate is completely exhausted. Growth ceases when aeration is halted, and the culture is cooled. A test for substrate is easily achieved by sampling. Cells are removed from samples by centrifugation, a small aliquot of supernatant is added to a solution of 50 m M NaOH, then the absorbance at 282 nm is measured. The concentration of p-hydroxybenzoate is calculated from its extinction at 282 nm (16.3 m M -1 cm-l). A yield of 5 to 6 g of cells (wet weight) per liter is obtained. The cells can be stored as a frozen paste at - 7 0 °. Procedure. The procedure below has been arranged for amounts up to 100 g wet weight of cells. However, scale-up for larger quantities should not be difficult. With organization, the procedure can be completed in 2 days. This time scale is recommended. On Day 1 cells are thawed with 2 volumes of extraction buffer (50 m M potassium phosphate, 0.5 m M EDTA, 0.5 m M p-hydroxybenzoate, pH 7.0), and 5 mg of deoxyribonuclease I is added. The slurry is sonicated on ice with the temperature maintained under 16 ° until enzyme activity reaches a maximum (usually within 3 min). Approximately 13,000 units of enzyme should be obtained from 100 g of cells (at least 200 mg of enzyme). After the addition of a further 2 volumes of cold extraction buffer, the mixture is centrifuged at 30,000 g for 30 min at 2 °. The yellow supernatant

[ 24]

HYDROXYBENZOATE HYDROXYLASE

141

is siphoned off, and solid a m m o n i u m sulfate is dissolved in it at the rate of 24.5 g per 100 ml. The pH is adjusted to 7.0 with ammonia, and the mixture is stirred until it reaches 15 °. Then the mixture is centrifuged at 30,000 g for 20 min at 15 °. From here, chromatographic steps are most easily run at room temperature without loss of enzyme. Hydrophobic chromatography. The supernatant is loaded onto a colu m n of DEAE-cellulose (Whatman DE-52, bed volume of 1.5 times the original cell weight converted to milliliters) equilibrated with running buffer (25 m M potassium phosphate, 1.0 m M EDTA, pH 7.0) containing 45% saturation of a m m o n i u m sulfate at room temperature (about 20°). The column is then washed with 2 volumes of the same solution, and fractions containing enzyme are eluted with running buffer containing 32% saturation of a m m o n i u m sulfate. With a slow flow rate, most of the enzyme is eluted in about 2 bed volumes. The enzyme fractions only are pooled, and protein is precipitated by adding solid a m m o n i u m sulfate to a final concentration of 75% saturation, with pH in the range of 6.5 to 7.0. The precipitate is recovered by centrifugation at 20,000 g for 10 min. The protein is dissolved in ice-cold affinity buffer (10 m M Tris-maleate, 0.3 m M EDTA, pH 7.0) by the addition of a volume of about 0.5 of cell weight. The solution is dialyzed against affinity buffer at 2 to 4°to remove a m m o n i u m sulfate (overnight). Affinity chromatography. On Day 2, the dialyzed solution is centrifuged to remove protein precipitate (after warming to 20°), then loaded onto a column of Blue Sepharose (Pharmacia Blue Sepharose CL-6B) equilibrated with affinity buffer at 20 ° (bed volume approximately the same as the original cell weight). The column is washed with 1 volume of affinity buffer. Protein is eluted with affinity buffer containing a gradient of 0 to 0.5 M KCI (gradient volume about 4 bed volumes). The fractions containing the enzyme peak are combined. Hydroxylapatite chromatography. The solution above is loaded directly onto a column of hydroxylapatite equilibrated with 7 m M potassium phosphate buffer, pH 7.0 at 20 ° (bed volume about 35% of the original cell mass, if the Bio-Rad product BioGel HT is used). The column is washed with 2 volumes of 8 m M potassium phosphate, pH 7.0, then the yellow enzyme product is eluted by flushing the column with 48 m M potassium phosphate, pH 7.0. Enzyme elutes in about 1 bed volume. Fractions containing enzyme with an A2so/A45oabsorbance ratio of less than 10 can be used for most experimental purposes. Yields of enzyme are in the range of 50 to 60% of the enzyme in the original cell-free extract. Crystallization. Crystallization may be used to obtain enzyme for analytical studies of the molecule. To the material from hydroxylapatite, EDTA is added to 1.0 raM. Solid a m m o n i u m sulfate is added slowly until

142

HYDROCARBONS AND RELATED COMPOUNDS

[24]

the solution is faintly turbid. The sample is quickly centrifuged, and the supernatant is allowed to stand in a cold room to slowly form crystals. Pure enzyme has an A2ao/A45o ratio of 8.5 and a specific activity of 62 to 64. This is equivalent to a molecular activity in the standard assay of 2900 to 3000 per minute, when an extinction value of 11.3 m M - t cm -t is used for the enzyme at 450 nm. Storage. Large quantities of enzyme can be stored indefinitely as a precipitate under a solution of 50 m M potassium phosphate and 0.5 m M EDTA, pH 6.5 to 7.0, with 70% saturated ammonium sulfate at 0 to 4 ° (away from light). Smaller quantities of enzyme can also be stored in solution (without ammonium sulfate) in the same buffer at - 7 0 ° (I>2 mg/ml). If enzyme has partly degraded for some reason, intact molecules can usually be purified to full specific activity by running the sample through a small column of hydroxylapatite as above. Properties Stability. A number of reports have appeared since the previous chapter in this series on the heterogeneity of preparations of enzyme from P. fluorescens. The definitive paper from van Berkel and M011ers showed that multiple forms of the enzyme are artifacts of purification and storage. A single cysteine residue (Cys-116) at the surface of the molecule is susceptible to oxidation by oxygen. As a result, sulfinate and sulfonate derivatives can form which confer charge heterogeneity on the molecule. In addition, weak covalent disulfide cross-links between molecules can occur, 9 resulting in higher order molecular aggregates which disrupt ordered crystalliTation of the enzyme. However, catalytic activity is not influenced by these changes. Van Berkel and Mfiller~ recommend a different final step in purification and storage of the enzyme from P.fluorescens with dithiothreitol to overcome these chemical changes. We have not used dithiothreitol in the storage of enzyme from P. aeruginosa as prepared by the method described here, although Cys-116 should be in the molecule, t° Even after long periods of storage in solution, the preparations of enzyme from P. aeruginosa showed only small amounts of oxidized enzyme molecules compared to the native molecule, as determined by ion-exchange high-performance liquid chromatography (HPLC). Van Berkel and Mfiller tt report that the enzyme has a tolerance to temperatures as high as 60°, but only in

s w. J. H. van Berkel and F. MOiler,Eur. J. Biochem. 167, 35 (1987). 9j. M. van der Laan, M. B. A. Swarte, H. Groendijk, W. G. J. Hol, and J. Drenth, Eur. J. Biochem. 179, 715 (1989). to B. Entsch, N. Yang, K. Weaich, and K. F. Scott, Gene71, 279 (1988). t t W. J. H. van Berkel and F. Mfiller, Eur. J. Biochem. 179, 307 (1989).

[24]

HYDROXYBENZOATE HYDROXYLASE

143

the pH range of 5.5 to 6.5. The enzyme can be used with impunity in the pH range 5 to 8 if the temperature is kept below 40 °. Molecular Properties. In solution at physiological concentrations, the native enzyme is a dimer of identical subunits 8 (with independent active sites) held together by noncovalent forces22 Each polypeptide is 394 amino acids long and contains one molecule of FAD) °,12 The native subunit molecular weight is 45,100 (from the known structure), but the dimer behaves as a molecule of 75 to 80,000 in solution, because of its hydrodynamic shape. 9 Owing to the efforts of Drenth's laboratory, there is now a large body of knowledge on the 3-dimensional structure of the enzyme from P. fluorescens, and this information is equally applicable to the enzyme from P. aeruginosa. 1°,~3 The complex between p-hydroxybenzoate and oxidized enzyme was first described at 0.25-nm resolution by Wierenga et al. 14Then the full amino acid sequence and its integration with the crystallographic data were reported in 1983.12 Since then, the structure has been refined to 0.19-nm resolution.15 A surprising discovery was that the tricyclic isoalloxazine ring is slightly twisted in the oxidized enzyme toward the configuration necessary to accommodate a flavin-C4a derivative. Other forms of the enzyme involved in catalysis have been reported at various resolutions: the complex between p-hydroxybenzoate and reduced enzyme, Is the complex between 3,4-dihydroxybenzoate and oxidized enzyme, ~6 enzyme without substrates, t7 and a model of flavin-C4a-peroxide in the active site with p-hydroxybenzoate)s In the crystalline state, the enzyme is a dimer, just as in free solution. This structural knowledge has greatly stimulated interest in further study of the enzyme. Substrate Dependence. There has not been much new information about range of substrates for this enzyme. There was a major report in 198019 on aromatic fluorine derivatives of p-hydroxybenzoate (reprints containing the correct methods can be obtained from Vincent Massey). t2 W. J, Weijer, J. Hofsteenge, J. J. Beintema, R. K. Wierenga, and J. Drenth, Eur. J. Biochem. 133, 109 (1983). ~3B. Entsch and D. P. Ballou, Biochim. Biophys. Acta 999, 313 (1989). ~4R. K. Wierenga, R. J. de Jong, K. H. Kalk, W. G. J. Hol, and J. Drenth, J. Mol. Biol. 131, 55 (1979). t5 H. A. Schreuder, P. A. J. Prick, R. K. Wierenga, G. Vriend, K. S. Wilson, W. G. H. Hol, and J. Drenth, J. Mol. Biol. 208, 679 (1989). ~6H. A. Schreuder, J. M. van der Laan, W. G. J. Hol, and J. Drenth, J. Mol. Biol. 199, 637 (1988). ,7 j. M. van der Laan, Doctoral dissertation, Groningen, 1986. ~s H. A. Schreuder, Doctoral dissertation, Groningen, 1988. ~9M. Husain, B. Entsch, D. P. Ballou, V. Massey, and P. J. Chapman, J. Biol. Chem. 255, 4189 (1980).

144

HYDROCARBONS AND RELATED COMPOUNDS

[24]

Fluorine can replace any or all of the ring hydrogens in p-hydroxybenzoate, and an effective substrate results. When fluorine is in the 3-position, it can be eliminated as fluoride. When this occurs, oxygenation of substrate becomes the rate-determining step. Other potentially important analogs of p-hydroxybenzoate are known: 2° 5-hydroxypicolinate is a powerful effector (i.e., it stimulates rapid oxidation of NADPH by the enzyme without being a substrate), whereas 4-aminosalicylate and 6-aminonicotinate are excellent competitive inhibitors which do not stimulate NADPH oxidation. Analogs of NADPH have generally not been useful in studying this enzyme. Structural analyses have shown that the enzyme does not have a conventional Rossman fold to bind NADPH. 2~ It has been reported on a number of occasions that some anions act as competitive inhibitors of NADPH binding. For example, CI-, I-, CNS-, and N3- are all effective inhibitors in millimolar concentrations. 22 However, the catalytic effect of these anions is much more complex than competitive inhibition. 5 Inorganic anions such as sulfate and phosphate are safe to use for enzyme kinetic studies. The first full description of the steady-state substrate kinetics of the enzyme from P. fluorescens was reported with p-mercaptobenzoate as substrate. :3 This has subsequently been followed by a full analysis with the native substrate, p-hydroxybenzoate, at 4 °,24 for comparison to studies of enzyme transient species in the reaction. A complete collection of steadystate parameters for the enzyme from P. aeruginosa at pH 8.0 (the pH optimum) and 250 has been publishedJ 3 The enzyme has a turnover number at these standard conditions of 3750 min-i. The kinetic parameters reported in this paper should be close to the actual values for the enzyme from P. fluorescens under standard conditions. Flavin Cofactor. As the component of the enzyme reaction at the center of catalysis, and the component readily studied in isolation (owing to electronic transitions in the visible), the flavin has received considerable attention. The perturbations of catalysis caused by analogs of the isoalloxazine ring have been studied by Massey's laboratory. Two important practical considerations surround this work. First, the natural nucleotide of the chemically modified riboflavin must be generated. The most favored method utilizes a preparation of flavokinase and FAD synthase from Brevibacterium ammoniagenes to generate the dinucleotide, as described 20 B. Entsch, unpublished observations (1975) and (1989). 21 R. K. Wierenga, J. Drenth, andG. E. Sehulz, J. Mol. Biol. 167, 725 (1983). 22 H. Shoun, K. Arima, and T. Beppu, J. Biochem. 93, 169 (1983). 23 B. Entsch, D. P. Ballou, M. Husain, and V. Massey, J. Biol. Chem. 251, 7367 (1976). 24 M. Husain and V. Massey, J. Biol. Chem. 254, 6657 (1979).

[24]

HYDROXYBENZOATE HYDROXYLASE

145

originally by Spencer et aL 25 Second, the natural FAD must be removed from the enzyme before binding the new analog. The simplest procedures involve dialysis or ammonium sulfate precipitation at low pH, as described in the papers by Ghisla et aL 26 and Entsch et aL 27 However, if extensive work in this area is planned, then it is worth investing in the procedure devised by Mtiller and van Berkel. 2s In this method, the enzyme is covalently adsorbed (presumably through Cys-l 16) to a column of AH-Sepharose substituted with a reactive thiol reagent. FAD is washed from the bound enzyme, and the apoprotein is eluted from the column with dithioerythritol. With this method, experience has shown that large amounts of apoprotein can be prepared with only modest loss of enzyme by denaturation (but the apoprotein loses activity with time2S). The final dialysis step described in the method can be eliminated if the only purpose is to bind a new FAD analog. It is only necessary to incubate the apoprotein with a 2-fold excess of the new FAD for a few minutes, then precipitate the enzyme with solid ammonium sulfate to 70% saturation, followed by molecular sieve chromatography (Sephadex G-25) to remove excess flavin and other solutes as required. Valuable information about the active site of the enzyme can be obtained from chemically modified FAD. For example, the chemical environment of the flavin can be tested by the incorporation of a reactive substituent, such as 7- and 8-halogen-substituted FAD 29 or 2-thio-FAD. 3° The reaction mechanism can be probed by appropriate changes to FAD. When 1-deaza-FAD was used as cofactor,27 the enzyme carded out each step in catalysis except the transfer of oxygen to p-hydroxybenzoate. The derivative, 6-hydroxy-FAD, yielded a competent enzyme which had a lower turnover rate than the native enzyme, a~ Subtle changes in catalysis with 6-hydroxy-FAD established the physical orientation of substrate to flavin during catalysis. A m i n o A c i d Changes. This enzyme, perhaps as much as any other enzyme, catalyzes a reaction under the absolute control of the protein involved. Thus, with the knowledge of structure available, there is considerable interest in probing the role of amino acid residues, particularly in the active site. In Holland, the principal investigators, van Berkel and 25R. Spencer,J. Fisher,and C. Walsh,Biochemistry 15, 1043(1976). 26S. Ghisla,B. Entsch,V. Massey,and M. Husain,Eur. J. Biochem. 76, 139 (1977). 27B. Entsch, M. Husain, D. P. Ballou, V. Massey,and C. Walsh,J. Biol. Chem. 255, 1420 (1980). 2sF. Miillerand W. J. H. van Berkel,Eur. J. Biochem. 128, 21 (1982). 29V. Massey,M. Husain,and P. Hemmerich,J. Biol. Chem. 255, 1393(1980). aoA. Claiborne,P. Hemmerich,V. Massey,and R. Lawton,J. Biol. Chem. 258, 5433 (1983). 3, B. Entsch, V. Massey,and A. Claiborne,J. Biol. Chem. 262, 6060 (1987).

146

HYDROCARBONS AND RELATED COMPOUNDS

[24]

MOiler, have made several studies of protein modification with reactive chemicals. They have, for example, attempted to target cysteine,a2 tyrosine,33 and arginine residues. ~ The problem, as usual, is the inability to rationally target active site residues by this approach. Alternative approaches include natural variations and mutations. As with the chemical approach, there is little hope of predictive modifications, or low probability of successful results. The techniques of gene manipulation hold much greater hope for the future of this workJ ° Although these methods are labor intensive, they are becoming easier to implement all the time. In my laboratory, tyrosine residues 201,222, and 385 (all in the active site of the enzyme) have been changed individually to phenylalanine. It was found that all three changes substantially modified the catalytic function of the enzyme in different aspects, without completely eliminating catalysis. 35 Thus, it should not be long before a picture of the protein function emerges. O x y g e n Reactions. The most comprehensive information on the oxygen reactions of flavoprotein oxygenases remains the 1976 paper by Entsch et al. 5 The great majority of the reaction model proposed at that time has been confirmed in other reports and investigations of other flavoproteins, mostly in association with Massey's laboratory. The enzyme studies have been supported by comprehensive chemical investigations of the initial reactions of appropriate reduced flavin models with oxygen.~ One great puzzle left from the results reported in 1976 was the nature of a transient chemical species found associated with the transfer of oxygen from flavin to the substrate, the crucial step in the formation of an oxygenated product. It was the injection of a novel physical technique into the study of this enzyme which may have solved the problem. Anderson et al. 37 used pulse radiolysis to study the interactions of flavins with oxygen. They have chemical evidence from reactions of p-hydroxybenzoate with hydroxyl radicals that the unknown enzyme intermediate is a radical pair between flavin and oxygenated product, which has some kinetic stability under some conditions with the enzyme.

32 W. J. H. van Berkel, W. J. Weijer, F. Mffller, P. A. Jekel, and J. J. Beintema, Eur. J. Biochem. 145, 245 (1984). 33 W. J. H. van Berkel, F. Miiller, P. A. Jekel, W. J. Weijer, H. A. Schreuder, and R. K. Wierenga, Eur. J. Biochem. 176, 449 (1988). R. A. Wijnands, F. Miiller, and A. J. W. G. Visser, Eur. J. Biochem. 163, 535 (1987). 35 B. Entsch, P. Bundock, and R. E. Wicks, Proc. Australian Biochem. Soc. 21, P54 (1989). T. C. Bruice, in "Flavins and Flavoproteins" (R. C. Bray, P. C. Engel, and S. G. Mayhew, eds.), p. 45. de Gruyter, Berlin and New York, 1984. 37 R. F. Anderson, K. B. Patel, and M. R. L. Stratford, J. Biol. Chem. 262, 17475 (1987).

[24]

H Y D R O X Y B E N Z O A THYDROXYLASE E

147

Genetics Further advances in our understanding of protein structure and function in this enzyme will include an application ofgene technology. It would thus be sensible to integrate this work if possible with genetics and bacterial gene control. The bacterium P. fluorescens has not been used as a model for bacterial genetics, but it does exist within a closely related group of bacteria known as the fluorescent pseudomonads, which includes favorite targets for genetic analysis, P. aeruginosa and P. putida.3S It has been found from the accurate sequence of the gene for p-hydroxybenzoate hydroxylase (pobA) that the enzyme from P. aeruginosa is the same as that from P. fluorescens except for two residues, amino acids 228 and 249. ~° Both side chains are at the surface of the enzyme, and the changes have very little influence on catalysis. ~3 Thus, the gene from P. aeruginosa is an ideal model to link the genetics of pseudomonads to detailed enzyme analysis. Only one gene has been identified for the metabolism ofp-hydroxybenzoate by microorganisms (pobA). There must be a permease-type mechanism for the uptake of this compound into cells, but no information exists on this subject. The product of the enzyme reaction (protocatechuate, or 3,4-dihydroxybenzoate) is considered an integral member of the central aromatic degration pathways, a9 The gene in P. aeruginosa is located about the 30' position in the chromosome and may be close to, but not directly linked with, some genes involved in the degradation ofprotocatechuate via the fl-ketoadipate pathway, as The gene is also located in the chromosome of P. putida. Earlier studies had shown that the enzyme is under tight control and is induced by its substrate, but only in the absence of some alternate carbon s o u r c e s . 39 Recently, cloned pobA from P. aeruginosa has been identified, sequenced, and structurally analyzed) ° It was found that the intact gene is not expressed in Escherichia coli, but expression is successful when the protein coding region is linked to an E. coli promoter. A successful strategy has now been developed35 to engineer site-specific changes in the enzyme through manipulations of the gene with synthetic oligonudeotides, according to the method of Kunkel et al. 4° The same techniques can be used for gene manipulation to investigate gene control in P. aeruginosa. It should be noted that similar work is now in progress in investigations of the sal gene for salicylate monooxygenase from P. cepacia. 4~ 3s B. 39 R. 4o T. 4t y.

W. Holloway and A. F. Morgan, Annu. Rev. Microbiol. 40, 79 (1986). Y. Stanier and L. N. Ornston, Adv. Microb. Physiol. 9, 89 (1973). A. Kunkel, J. D. Roberts, and R. A. Zakour, this series, Vol. 154, p. 367. Kim and S.-C. Tu, Arch. Biochem. Biophys. 269, 295 (1989).