[81] Aldehyde dehydrogenases from Pseudomonas aeruginosa

484

OXIDATION--REDUCTION ENZYMES

[81 ] A l d e h y d e

Dehydrogenases

from Pseuclomonas

[81]

aeruginosa

B y J. P. VANDECASTEELE and L. GUERRILLOT

CHs(CH2)nCHO + NAD(P+) + H20 ~ CHa(CH2)nCOOH + NAD(P)H + H + Several specific aldehyde dehydrogenases, such as a-ketoglutarate semialdehyde dehydrogenase ~ and succinate semialdehyde dehydrogenase, z have been described in P s e u d o m o n a s . In P s e u d o m o n a s aeruginosa an aldehyde dehydrogenase induced by growth on ethanol has also been studied. 3 The two enzymes described below, which are noninducible soluble e n z y m e s from P. aeruginosa and utilize a wide range of aliphatic aldehydes, have been studied by Guerrillot and Vandecasteele. 4 Another different aldehyde dehydrogenase with a wide specificity has been reported in P s e u d o m o n a s fluorescens. 5

NAD+-Linked Aldehyde Dehydrogenase 4 (EC 1.2.1.3)

Assay Method Principle. The reduction of N A D + is measured at 30 ° with a recording s p e c t r o p h o t o m e t e r at 340 nm. Reagents

Butanal (from Fluka) 0.21 m M dissolved in potassium p y r o s p h a t e buffer 54 m M , p H 8.6. Because of the limited solubility of aldehydes, butanal or any other aldehyde assayed is dissolved in the buffer at a concentration close to the final assay concentration. In these conditions, aldehydes are present as true solutions in the buffer to avoid the artifacts resulting from the use o f emulsions, as reported in the case of long-chain alcohols. 6 N A D ÷, 30 m M E. Adams and G. Rosso, J. Biol. Chem. 242, 1802 (1967). 2 D. M. Callewaert, M. S. Rosemblatt, K. Suzuki, and T. T. Tchen,J. Biol. Chem. 248, 6009 (1973). 3 R. G. von Tigerstr6m and W. E. Razzel, J. Biol. Chem. 243, 6495 (1968). 4 L. Guerrillot and J. P. Vandecasteele, Eur. J. Biochem. 81, 185 (1977). 5 W. B. Jakoby, J. Biol. Chem. 232, 89 (1958). 6 j. p. Tassin and J. P. Vandecasteele, C. R. Hebd. Seances Acad. Sci. Ser. D 272, 1024 (1971).

METHODS IN ENZYMOLOGY, VOL. 89

Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181989-2

[81]

ALDEHYDEDEHYDROGENASESFROMP. aeruginosa

485

Procedure. To the cuvettes (10 mm light path) are added 2.8 ml of the butanal solution in buffer, 0.1 ml of NAD ÷, water, and enzyme to a final volume of 3 ml. (Final concentrations are 2 mM for butanal, 50 mM for potassium pyrophosphate and 1 mM for NAD+.) The reaction is initiated by addition of enzyme. Unit. Enzymic activities are expressed in enzyme units. One unit is defined as the amount that transforms 1 /zmol of substrate per minute. Protein Assay. A modified Folin method is used to avoid interfering reactions between reagent and SH groups that are present in elution buffers. Typically, 50-150 txg of protein in 0.5 ml are precipitated by 2 ml of 25% trichloroacetic acid. This preparation is kept for 15 min at 0° before centrifugation for 15 min at 10,000g at 2°. The pellet is resuspended in 0.5 ml of NaOH, and then the assay is performed by the method of Lowry et al. 7

Purification Procedure Step 1. Growth o f the Microorganism. The strain used, Pseudomonas aeruginosa 196 Aa, isolated by Traxler and Bernard, 8 is grown in sterile

conditions in a 12-liter fermentor (Magnaferm, from New Brunswick). The salts medium used 9 contains per liter: NH4C1, 2 g; KH2PO4, 4 g; Na2HPO4 • 12 H~O, 6 g; Mg SO4 • 7 H20, 0.3 g; FeSO4 • 7 H20, 10 mg; ZnSO4"7 H20, 0.1 mg; CUSO4"5 H20, 0.1 mg; HzBOz, 40 /.tg; MnSO4" H20, 22 ~g; MoTO~a(NH4)6' 4 H20, 153 /zg. This medium is adjusted to pH 7.0 by 5 N KOH and sterilized 25 min at 120°. The carbon source is 2% (w/v) glucose (final concentration). It is added to the sterile salts medium as a concentrated (40% w/v) solution that has been sterilized separately (20 min at 105°). The culture is performed at 30° with stirring (1000 rpm) and forced aeration (0.5 liter liter -1 min-l). The pH is regulated at 7.0 by 4 N NH4OH. Cells are harvested in the exponential phase of growth (about 9 hr after inoculation) by centrifugation, washed with 10 mM phosphate buffer (pH 7.1), and kept at - 3 0 ° until used. Step 2. Preparation o f Extracts. Cells (80 g wet weight) are suspended by portions of 10 g in 200 ml of potassium phosphate buffer (pH 7.2) containing 7 mM 2-mercaptoethanol and 1 mM dithiothreitol; 1.6 mg of DNase (Sigma, DHC) and 8 ml of 0.2 M magnesium sulfate are added. The cell suspensions, immersed in an ice bath, are disintegrated by sonication 70. H. Lowry, N. J. Rosebrough,A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

R. W. Traxler and J. M. Bernard,Int. Biodeterior. Bull. 5, 21 (1969). 9 D. S. Robinson,Antonie Van Leeuwenhoek J. Microbiol. Serol. 30, 303 (1964).

486

OXIDATION-REDUCTION ENZYMES

,o =

f I

[81]

-'"

I

5

Z" I-

40 e~ ibo,~o~oFraction number

FlG. 1. Separation of the NAD+-dependent and NADP+-dependent aldehyde dehydrogenases by DEAE-cellulose chromatography. A 38 mm x 370 mm column is used. The elution rate is 95 ml hr -z, and fractions of 10 ml are collected. & &, NADP+-dependent aldehyde dehydrogenase activity; O O, NAD+-dependent aldehyde dehydrogenase activity; [] [], protein (mg x ml-1); . . . . , concentration of elution phosphate buffer (pH 7.8). Protein concentration in fraction 140 is 22 mg m1-1. Adapted from Guerrillot and Vandecasteele,4 with permission.

(six periods of 15-20 sec, to prevent a temperature increase above 12°). Then the pooled preparations are centrifuged at 3° for 60 min at 100,000g. Step 3. Protamine Sulfate Treatment. A 2% protamine sulfate (7.2 ml) solution is added dropwise to the extract (138 ml), which is stirred for 30 min and then centrifuged for 20 rain at 48,000 g. From this step on, all enzyme preparations and chromatography or dialysis buffers (unless otherwise stated) contain 16% glycerol and either 7 mM 2-mercaptoethanol or 1 mM dithiothreitol (final concentrations) to maintain sufficient stability (see Properties). The protamine sulfate supernatant is dialyzed against 20 mM phosphate buffer pH 7.3. Step 4. DEAE-Cellulose Chromatography. The dialyzed extract (178 ml) adjusted to pH 7.8 is adsorbed on the top of a DEAE-cellulose column previously equilibrated with 20 mM phosphate buffer (pH 7.8) and eluted with a concentration gradient of the phosphate buffer (pH 7.8) as shown in the elution diagram (Fig. 1). This gradient is obtained with a programmable gradient pump system [Dialagrad from Instrument Specialties Co., Lincoln, Nebraska (Isco)]. A slightly convex gradient with similar initial and final concentrations can also be used and is obtained with a classical two-vessels device, presented earlier in this series. ~° As shown in Fig. 1, separation of the two aldehyde dehydrogenase takes place on this column. 10 E. A. Peterson and H. A. Sober, this series, Vol. 5 [1].

[81]

ALDEHYDE DEHYDROGENASES FROM

487

P. aeruginosa

TABLE I PURIFICATION OF NAD-DEPENDENT ALDEHYDE DEHYDROGENASE u'b

Preparation Supernatant, 100,000 g Protamine s u p e r n a t a n t Dialyzed s u p e r n a t a n t DEAE-cellulose eluate (fractions 95 -120) Hydroxyapatite eluate Sephadex G-200 eluate (173-240 ml) Fraction (198-203 ml) of Sephadex G-200

Volume (ml) 138 142 178

Protein (mg/ml) 44 44.8 36

Total activity (units)

Specific activity (units/mg protein)

Yield (%)

Purification (fold)

830 850 710

0.136 0.134 0.112

--100

--1

0.57 3.4

40 20

5.1 30

254 232

1.9 0.18

280 140

67

0.099

100

5

0.11

11

15.2 20

14 --

135 178

" A d a p t e d from Guerrillot and Vandecasteele, 4 with permission. b Activity determinations are performed in 50 m M KzHP207 (pH 8.6) with 0.2 m M butanal.

In spite of various attempts, however, complete separation has not been achieved in this step. From this step on, purification of the two enzymes is carried out independently. Fractions 95-120 are pooled together and then dialyzed against 20 mM phosphate buffer (pH 6.8) (Table I). Step 5. Hydroxyapatite Chromatography. The preparation is concentrated to a volume of 39.5 ml by ultrafiltration (Amicon, UM-20E membrane) and subjected to chromatography on a hydroxyapatite column (25 × 340 nm) previously equilibrated with 20 mM phosphate buffer (pH 6.8). Elution, performed at a flow rate of 30 ml/hr, is started with the same buffer for 250 ml, then continued with a concentration gradient of the phosphate buffer (pH 6.8), obtained by the methods described in step 4, the phosphate concentration increasing from 20 mM at 250 ml to 70 mM at 1000 ml of elution volume. Step 6. Sephadex G-200 Chromatography. The preparation is then adjusted to 100 mM potassium phosphate (pH 7.1). The Sephadex column (25 x 780 mm) is equilibrated and eluted (flow rate 8 ml/hr) with the same phosphate buffer. The purest preparation (eluate between 198 and 203 ml) has a specific activity of 20 units per milligram of protein. Electrophoresis of this preparation on polyacrylamide gel allows a rough estimation of enzyme purity of about 20%. Typical results of the various purification steps are summarized in Table I.

488

OXIDATION--REDUCTION ENZYMES

[81]

Properties

Molecular Weight. A molecular weight of 225,000 _+ 15,000 has been determined by gel filtration on Sephadex G-200. Apparent Kinetic Constants. Aliphatic aldehydes in a range from C2 to at least C10 are used by this enzyme. Kinetic constants for these aldehydes have been determined in 50 mM potassium pyrophosphate (pH 8.6) in the presence of 1 mM NAD +. Km values decrease with increasing aldehyde chain length from 165 ~ M for ethanal, 45 ~M for butanal, 9.5 /zM for hexanal, and 1.3 ~M for decanal, whereas variations of V are small. In the same buffer, an apparent Km value for NAD + of 44 ~ M is found in the presence of 200/zM butanal. Influence of pH, Buffer, and Ions on Activity. Small variations of Km values for aldehydes with pH and buffer composition are observed. In Tris buffer, however, a much higher Km value is found, possibly because of the formation of imine bonds between Tris and aldehyde. V values for aldehydes somewhat increase with pH. Potassium and ammonium ions have a slight activation effect on the enzyme. Enzyme Stability. Glycerol and reducing agents have a clear stabilization effect on enzyme activity. Dithiothreitol or 2-mercaptoethanol can also partially reactivate the enzyme that has been inactivated by storage in the absence of reducing agents. Alkaline pH values also inactivate the enzyme. At pH 7.4 the enzyme is stable, but at pH 8.6 (in 50 mM potassium pyrophosphate) 90% activity is lost at 30° in 10 min. In the presence of 1 mM NAD +, however, no inactivation takes place under the same conditions. As a consequence, the enzyme is routinely stored and handled in 50 mM potassium phosphate, pH 7.4, containing 16% glycerol and 1 mM dithiothreitol. At - 8 ° under these conditions the purified enzyme loses 25% activity in 6 months. Regulation of Enzyme Synthesis. Very similar enzyme activities are found in high-speed supernatants of extracts of cells grown on glucose, succinate, malonate, n-heptane, or n-hexadecane. The enzyme is soluble; very little activity is observed in the particulate fraction of the extracts except when hydrocarbons are used as carbon sources. Growth on hydrocarbon induces the synthesis of a specific membrane-bound NAD ÷dependent aldehyde dehydrogenase. Kinetic Studies. Detailed steady-state kinetic studies including bireactant initial velocity and product inhibition experiments have been performed. H The results indicate the existence of a sequential mechanism. 11L. Guerrillot, "Contribution/tl'6tude du m6tabolismedes hydrocarbureschez les microorganismes. Etude des ald6hyded6shydrog6nasesde Pseudomonas aeruginosa." Doctoral Thesis No. 2013, Universityof Paris-Sud-Orsay, 1978.

[81]

489

ALDEHYDEDEHYDROGENASESFROMP. aeruginosa TABLE II PURIFICATIONOF NADP-DEPENDENTALDEHYDEDEHYDROGENASEa'b

Preparation Supernatant, 100,000 g Protamine supernatant Dialyzed supernatant DEAE-cellulose eluate (fractions 80-94) Hydroxyapatite eluate Sephadex G-200 eluate (175-235 ml) Fraction (200-205 ml) of Sephadex G-200

Volume (ml) 138 142 178

Specific Total activity PurifiProtein activity (units/rag Y i e l d cation (mg/ml) (units) protein) (%) (fold) 44 44.8 36

850 830 650.

0.14 0.13 0.10

--100

--1

148 170

1.5 0.065

310 180

1.31 17.0

48 28

12.8 166

60

0.08

156

32.0

23.5

314

5

0.09

16.7

37.0

363

a Adapted from Guerrillot and Vandecasteele,4 with permission, b Activity determinations are performed in 50 mM K3HP207 (pH 8.6) with 4 mM pentanal. The product inhibition pattern suggests an ordered pathway, but its complexity precludes a simple interpretation. N A D P + - D e p e n d e n t Aldehyde Dehydrogenase 4 (EC 1.2.1.4) Assay Method Assay of enzyme activity is performed as described for the N A D ÷dependent e n z y m e except that the reagents used are 4.3 m M pentanal (from Fluka) dissolved in 54 m M potassium pyrophosphate buffer, p H 8.6, and 15 m M N A D P ÷. The final concentrations in the assay are 4 m M for pentanal, 50 m M for potassium pyrophosphate, and 0.5 m M for N A D P +. Purification Procedure Steps 1-4 which are the same as for the NAD+-dependent e n z y m e have been described above. Fractions 80-94 of the c h r o m a t o g r a p h y on DEAE-cellulose (Fig. 1), which contain the N A D P ÷ - d e p e n d e n t activity, are further purified by c h r o m a t o g r a p h y on hydroxyapatite (step 5) and on Sephadex G-200 (step 6) using the same conditions as for the N A D ÷dependent enzyme. The results o f the complete purification procedure are given in Table II. The purest fraction of the Sephadex G-200 eluate (200-

490

O X I D A T I O N - - R E D U C TENZYMES ION

181]

205 ml) is 90-95% pure from gel electrophoresis determinations. It contains no NAD÷-dependent activity. Properties

Molecular Weight. A molecular weight of 215,000 _+ 15,000 has been obtained by gel filtration on Sephadex G-200. Apparent Kinetic Constants. Kinetic constants have been determined in 50 mM potassium pyrophosphate, pH 8.6, for aldehyde chain lengths ranging from C2 to C14, in the presence of 0.5 mM NADP ÷. Km values decrease with increasing aldehyde chain lengths from 35,000 /xM for ethanal to 435 /xM for pentanal, 250 /~M for hexanal and 15 /~M for tetradecanal. V values present two maxima for pentanal and for decanal. In the same buffer, an apparent Km value for NADP + of 65 tzM is found in the presence of 4 mM pentanal. Influence of pH, Buffer, Ions on Activity. Km and V values for aldehydes increase with pH in phosphate, pyrophosphate, or glycine-KOH buffers. Tris-HC1 cannot be used, as very low activities are observed in this buffer. Strong enhancement of enzyme activity by alkaline ions, in particular by potassium ions, are observed. Enzyme Stability. The protective effect of glycerol as well as the stabilization and reactivation by thiols are also observed for this enzyme. Inactivation at alkaline pH values, however, does not take place in this case. The NADP÷-dependent enzyme is handled and stored in the same medium as the NAD÷-dependent enzyme. At - 8 °, there is a 12% loss in activity in 6 months under these conditions. Regulation of Enzyme Synthesis. The NADP÷-dependent enzyme is soluble. Cell-free extracts from cells grown on the carbon sources mentioned above contain quite similar activities. Extracts from ethanol-grown cells, however, contain an additional soluble NADP÷-dependent aldehyde dehydrogenase that has not been characterized in detail. Kinetic Studies. The same studies have been performed as for the NAD+-dependent enzyme, 1~ and the results, although different in some respects, support the same general conclusions.