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Biochem. J. (1988) 255, 653-661 (Printed in Great Britain) Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus Substrate specificities and inhibition studies Robert W. MAcKINTOSH* 4nd Charles A. FEWSONt Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K. The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan- 1-al and octan- 1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 1O/,M. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzalde- hyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular, ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potent inhibitors of the enzyme. INTRODUCTION Acinetobacter calcoaceticus N.C.I.B. 8250 can grow on both benzyl alcohol and benzaldehyde, which are oxidized to benzoate by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase LI. These two enzymes have been purified (MacKintosh & Fewson, 1988) and their characterization forms part of an overall com- parison of the soluble NAD+-dependent alcohol dehydro- genases and aldehyde dehydrogenases that are present in A. calcoaceticus and in other micro-organisms (Mac- Kintosh & Fewson, 1987a). In the present paper we describe the substrate specificities of the two enzymes and the effects of several classes of inhibitors on their activities. The results are related to the metabolism of aromatic compounds by A. calcoaceticus. In addition, the kinetic properties of the enzymes are compared with those of some well-characterized eukaryotic alcohol dehydrogenases and aldehyde dehydrogenases. Preliminary accounts of parts of this work have been published (MacKintosh & Fewson, 1987a,b). EXPERIMENTAL Materials Chemicals were of the best quality commercially available and most of them were obtained from the sources described by MacKintosh & Fewson (1988). Yeast aldehyde dehydrogenase (K+-activated) and horse liver alcohol dehydrogenase were obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). Bacteria Acinetobacter calcoaceticus N.C.I.B. 8250, the wild- type strain, was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland, U.K. Stock cultures were maintained as described previously (Allison et al., 1985), and large amounts of bacteria for enzyme purification were grown as described by MacKintosh & Fewson (1988). Vol. 255 * Present address: Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, U.K. t To whom correspondence and reprint requests should be addressed. 653

Transcript of from Acinetobacter calcoaceticus

Page 1: from Acinetobacter calcoaceticus

Biochem. J. (1988) 255, 653-661 (Printed in Great Britain)

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II

from Acinetobacter calcoaceticusSubstrate specificities and inhibition studies

Robert W. MAcKINTOSH* 4nd Charles A. FEWSONtDepartment of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.

The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehydedehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydesand the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was themost effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second mosteffective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcoholdehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being bettersubstrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehydewas much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single smallsubstituent group in the meta or para position were better substrates than any other benzaldehydederivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan- 1-al andoctan- 1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde whenthe assay concentration exceeded approx. 1O/,M. Benzaldehyde dehydrogenase II, but not benzyl alcoholdehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcoholdehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagentsiodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzalde-hyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzylalcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agentsEDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausiblemetabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP.Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular,ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potentinhibitors of the enzyme.

INTRODUCTION

Acinetobacter calcoaceticus N.C.I.B. 8250 can grow onboth benzyl alcohol and benzaldehyde, which areoxidized to benzoate by benzyl alcohol dehydrogenaseand benzaldehyde dehydrogenase LI. These two enzymeshave been purified (MacKintosh & Fewson, 1988) andtheir characterization forms part of an overall com-parison ofthe soluble NAD+-dependent alcohol dehydro-genases and aldehyde dehydrogenases that are present inA. calcoaceticus and in other micro-organisms (Mac-Kintosh & Fewson, 1987a). In the present paper wedescribe the substrate specificities ofthe two enzymes andthe effects of several classes of inhibitors on theiractivities. The results are related to the metabolism ofaromatic compounds by A. calcoaceticus. In addition,the kinetic properties of the enzymes are compared withthose of some well-characterized eukaryotic alcoholdehydrogenases and aldehyde dehydrogenases.

Preliminary accounts of parts of this work have beenpublished (MacKintosh & Fewson, 1987a,b).

EXPERIMENTALMaterials

Chemicals were of the best quality commerciallyavailable and most of them were obtained from thesources described by MacKintosh & Fewson (1988).Yeast aldehyde dehydrogenase (K+-activated) and horseliver alcohol dehydrogenase were obtained from SigmaChemical Co. (Poole, Dorset, U.K.).

BacteriaAcinetobacter calcoaceticus N.C.I.B. 8250, the wild-

type strain, was obtained from the National Collectionof Industrial Bacteria, Aberdeen, Scotland, U.K. Stockcultures were maintained as described previously (Allisonet al., 1985), and large amounts of bacteria for enzymepurification were grown as described by MacKintosh &Fewson (1988).

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* Present address: Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, U.K.t To whom correspondence and reprint requests should be addressed.

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Enzyme purificationBenzyl alcohol dehydrogenase and benzaldehyde

dehydrogenase II were purified from A. calcoaceticusN.C.I.B. 8250 as described by MacKintosh & Fewson(1988).

Measurement of enzyme activityThe activities of purified benzyl alcohol dehydrogenase

and benzaldehyde dehydrogenase II were measured bymonitoring the rates of reduction of NAD+ as describedby MacKintosh & Fewson (1988).

Absorption coefficients and correction factors used in thecalculation of enzyme activitiesSome of the aromatic substrates and products of

benzyl alcohol dehydrogenase and benzaldehyde de-hydrogenase II absorb at 340 nm, and so it was necessaryto determine correction factors for the initial ratesobserved in the presence of these compounds. Theabsorption coefficients were therefore determined underthe same conditions as were used for the enzyme assays.Table 1 shows the substrates and products of benzylalcohol dehydrogenase and benzaldehyde dehydrogenaseII that have absorption coefficients of more than 1 00 ofthe value of NADH at 340 nm. All other substrates andproducts have molar absorption coefficients that are lessthan 0.063 x I03 M-l cm-1 at 340 nm (i.e. below 10 ofthe absorption coefficient of NADH). The measuredchanges in absorbance were corrected where necessary togive true estimates of the rates of formation of NADH.Correction factors were calculated in the followingway: 6

Correction factor NADH(6NADH _substrate +Cproduct)

Coniferaldehyde was not commercially available andso its absorbance at 340 nm could not be determined.However, the ability of benzyl alcohol dehydrogenase tooxidize coniferyl alcohol was monitored at 400 nm byfollowing the accumulation with time of coniferaldehyde,which has an absorption coefficient at 400 nm (pH 9.2)of approx. 22.4 x 103 M-1 * cm-1 (Jaeger et al., 1981). Theabsorption coefficient of coniferyl alcohol at 400 nm(pH 9.2) was found to be only 0.04 x 103 M-l cm-l.The standard assay procedure was modified by thereplacement of benzyl alcohol by coniferyl alcohol andthe omission of hydrazine.

Determination of kinetic parametersTo obtain Km and maximum-velocity values for the

substrates ofbenzyl alcohol dehydrogenase and benzalde-hyde dehydrogenase II, initial velocities were measuredat several non-saturating concentrations of each sub-strate. Initial velocities were analysed by using theEnzpack computer program (Williams, 1985), which willdetermine the kinetic coefficients by the direct-linearmethod (Eisenthal & Cornish-Bowden, 1974). ApparentMichaelis constants and maximum velocities were ob-tained for substrates (alcohols and aldehydes) at a fixedconcentration of cofactor. For double-reciprocal plotseach initial-velocity determination was done in duplicate.The concentrations of stock solutions of benzyl alcohol,benzaldehyde, NAD+ and NADH were standardized asdescribed by MacKintosh & Fewson (1988).

Table 1. Molar absorption coefficients of aromatic substratesand products of benzyl alcohol dehydrogenase andbenzaldehyde dehydrogenase II

The c340 values were measured under two different sets ofconditions.

10-3 x 6340

Compound (M-1. cm-1)

(a) With the benzyl alcohol dehydrogenase assay bufferCinnamaldehyde 2.003,4-Dimethoxybenzaldehyde 0.162-Hydroxybenzaldehyde 0.703-Hydroxybenzaldehyde 0.613-Hydroxybenzyl alcohol 0.174-Hydroxybenzaldehyde 0.204-Hydroxy-3-methoxybenzaldehyde 5.604-Hydroxy-3-methoxybenzyl alcohol 0.073-Methoxybenzaldehyde 0.643-Methoxybenzyl alcohol 0.214-Methoxybenzaldehyde 1.304-Methoxybenzyl alcohol 0.23

(b) With the benzaldehyde dehydrogenase II assay bufferCinnamaldehyde 1.432-Hydroxybenzaldehyde 2.822-Hydroxybenzoic acid 0.193-Hydroxybenzaldehyde 1.763-Hydroxybenzoic acid 1.264-Hydroxybenzaldehyde 22.133-Methoxybenzaldehyde 0.55Thiophen-2-carboxaldehyde 0.09

Esterase activityEsterase activity was measured as described by Ting &

Crabbe (1983). The reaction mixture (3 ml) contained2.0 ml of 75 mM-sodium pyrophosphate buffer, pH 7.4 or8.4 (50 mm assay concentration), enzyme, and 0.05 ml of30 mM-4-nitrophenyl acetate in acetone (0.5 mm assayconcentration) to initiate the reaction. The rate ofappearance of 4-nitrophenol was monitored at 400 nm.The molar absorption coefficient of 4-nitrophenol is9.8 x 10'0 '- cm-1 at pH 7.4 and 18.3 x103 M-'lcm-' atpH 8.4 (Ting & Crabbe, 1983).

Analytical methodsProtein concentrations were determined by the pro-

cedure of Bradford (1976). Bovine serum albumin wasused as the standard and it was assumed that a solutioncontaining 1 mg/ml had an A280 of 0.65 (Janatova et al.,1968).

Preparation of buffersBuffers were prepared at room temperature by adjusting

the pH values of approximately five-fourths strengthsolutions with HCl, NaOH or KOH and then making tovolume.

RESULTS AND DISCUSSION

Substrate specificityAn extensive variety of aliphatic and aromatic alcohols

and aldehydes was tested as potential substrates forbenzyl alcohol dehydrogenase and benzaldehyde de-hydrogenase II. Some of the compounds tested were notoxidized at significant rates, and Table 2 lists the

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Table 2. Alcohols and aldehydes that were not substrates of benzyl alcohol dehydrogenase or benzaldehyde dehydrogenase II

Benzyl alcohol dehydrogenase activity was assayed with 2 mM-NAD+ and 0.2 mm alcohol. Benzaldehyde dehydrogenase IIactivity was assayed with 2 mM-NADI and both 0.01 mm and 0.1 mM aldehyde. The following compounds were oxidized at< 0.50% of the rate observed with benzyl alcohol or benzaldehyde respectively.

(a) Alcohols that were not oxidized by benzyl alcohol dehydrogenaseAromatic alcohols:

2-Bromobenzyl alcohol, DL-l-phenylethanol, 2-phenylethanolOther alcohols:

Allyl alcohol, butan-l-ol, butan-2-ol, cis-cyclohexane-1,2-diol, trans-cyclohexane-1,2-diol, cyclohexanol, decan-l-ol, ethanol,heptan-l-ol, hexan-l-ol, hexahydrobenzyl alcohol, inositol, mannitol, methanol, octan-l-ol, pentan-2-ol, propan-l-ol,propan-2-ol, pyridine-3-methanol, pyridine-4-methanol, sorbitol

(b) Aldehydes that were not oxidized by benzaldehyde dehydrogenase IIAromatic aldehydes:

2-Bromobenzaldehyde, 2-chlorobenzaldehyde, 2,6-dichlorobenzaldehyde, 2,5-dihydroxybenzaldehyde, 3,4-dihydroxy-benzaldehyde, 2,4-dimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 2-fluorobenzaldehyde, 4-hydroxy-3-methoxy-benzaldehyde, 4-isopropylbenzaldehyde, 2-methoxybenzaldehyde, 2-methylbenzaldehyde, 3-methylbenzaldehyde, pentafluoro-benzaldehyde, phenylacetaldehyde

Aliphatic aldehydes:Acetaldehyde, butyraldehyde, decylaldehyde, formaldehyde, propionaldehyde

compounds that were not oxidized at above 0.5 %0 of therate observed with benzyl alcohol or benzaldehyderespectively. Table 3 records the kir.etic coefficients ofa range of alcohols and aldehydes that were oxidizedby benzyl alcohol dehydrogenase and benzaldehydedehydrogenase II. The turnover numbers, kcat, weredetermined from the apparent maximum-velocity valuesand this allowed the calculation of the specificityconstants, kcat /apparent Km. It is usually considered thatthe higher the specificity constant the closer the structureof the substrate transition-state intermediate is to theenzyme's active site (Fersht, 1985). Benzyl alcohol andbenzaldehyde have the highest specificity constantswith benzyl alcohol dehydrogenase and benzaldehydedehydrogenase II respectively, and the acceptability ofmany of the other substrates appears to depend on theposition and size of the substituent groups on thearomatic ring.

Benzyl alcohol dehydrogenase is in general specific foraromatic alcohols, although perillyl alcohol, a cyclohex-1-ene compound, is also a good substrate. The benzenering can be replaced by a thiophen or furan ring, butapparently not by a pyridine ring. There appears to be apreference for aromatic alcohols with small substituentgroups, preferably away from the reactive carbinolgroup, i.e. at the para rather than the ortho position; 2-bromobenzyl alcohol was the only substituted benzylalcohol tested that was not oxidized. This suggests thatthe active site of the enzyme may be a cleft structure,substituents in the para position on the aromatic ringbeing least sterically and electrostatically hindered forbinding. The electron-withdrawing properties of thesubstituent groups on the aromatic ring may also beinvolved in dictating the acceptability of a particularsubstrate (Klinman, 1972). Cinnamyl alcohol and coni-feryl alcohol, which have an alkenyl group between thereactive carbinol and the aromatic ring, were oxidized bybenzyl alcohol dehydrogenase. In the trans configurationthe carbinol groups of these two alcohols may becorrectly positioned for reactivity within the active site.2-Phenylethanol, on the other hand, does not have analkenyl group near the carbinol and was not a substrate.

The alcohol specificity of A. calcoaceticus benzylalcohol dehydrogenase resembles that of some otherbacterial aromatic alcohol dehydrogenases, such as thebenzyl alcohol dehydrogenase (Suhara et al., 1969) andperillyl alcohol dehydrogenase (Ballal et al., 1966) fromPseudomonas spp. and the coniferyl alcohol dehydro-genase from Rhodococcus erythropolis (Jaeger et al.,1981). Benzyl alcohol and perillyl alcohol gave thehighest kcat/apparent Km values with benzyl alcoholdehydrogenase from A. calcoaceticus, and this along withother shared properties suggests a similarity between thisenzyme and the benzyl alcohol dehydrogenase purifiedfrom toluene-grown Pseudomonas putida T-2 (Suharaet al., 1969), which oxidizes cyclohex- 1-ene ring com-pounds (e.g. perillyl alcohol) as well as aromatic alcohols.

Horse liver alcohol dehydrogenase has an exceptionallybroad substrate specificity and will oxidize both aliphaticand aromatic alcohols; benzyl alcohol is oxidized atapproximately the same rate as ethanol (Sund & Theorell,1963). The classical fermentative yeast alcohol dehydro-genase oxidizes a much smaller number of substrates,chiefly straight-chain alcohols. Benzyl alcohol is a verypoor substrate for yeast alcohol dehydrogenase, althoughcinnamyl alcohol is oxidized at almost 40 0 of the ratefor ethanol (Sund & Theorell, 1963). It has been shownthat the active site of yeast alcohol dehydrogenasecontains bulkier residues than does the active site of thehorse liver enzyme, and this may explain the morerestricted substrate specificity of the yeast enzyme(J6rnvall et al., 1978).

Benzaldehyde dehydrogenase II appears to have amuch more restrictive active site than does benzyl alcoholdehydrogenase. Aromatic (benzene, furan, pyridine orthiophen rings) aldehydes are preferred substrates,although hexan- 1-al and octan-1-al are also oxidized.Cinnamaldehyde, with its propen- 1-al group, and peril-laldehyde, which is non-planar and has a bulky iso-propenyl group, were also relatively poor substrates,although the former had the higher specificity constant.Aromatic aldehydes with more than one substituentgroup were not oxidized. However, aromatic aldehydeswith only one substituent group were substrates, and the

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Table 3. Kinetic coefficients of substrates for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II

Benzyl alcohol dehydrogenase (9.6 ng of protein/ml of assay mixture) and benzaldehyde dehydrogenase II (40 ng of protein/ml of assay mixture) activities were measured as described in the Experimental section. The initial velocities were calculated byapplication of the appropriate correction factors where necessary (see the text). The NADI concentration was fixed at 2 mM,and the substrate concentrations ranged in most cases from 0.01 to 0.1 mm for the alcohols, and from 0.001 to 0.2 mm for thealdehydes. The kcat values were calculated as (apparent maximum velocity)/(concentration of active sites) assuming subunitMr values of 39700 and 55000 respectively (MacKintosh & Fewson, 1988) and one active site per subunit.

SpecificityApparent Vm constant

Apparent Km (units/mg of kcat kcat/apparent KmSubstrate (#M) protein) (sj') (s-1./M-1)

(a) Benzyl alcohol dehydrogenaseBenzyl alcoholPerillyl alcohol4-Methoxybenzyl alcohol4-Hydroxybenzyl alcoholCinnamyl alcohol4-Isopropylbenzyl alcohol4-Hydroxy-3-methoxybenzyl alcoholConiferyl alcohol3,4-Dimethoxybenzyl alcohol3-Methoxybenzyl alcohol2-Hydroxybenzyl alcoholThiophen-2-methanol2-Methoxybenzyl alcoholFuran-2-methanol

(b) Benzaldehyde dehydrogenase IIBenzaldehyde4-Fluorobenzaldehyde3-FluorobenzaldehydeThiophen-2-carboxyaldehyde3-MethoxybenzaldehydePyridine-4-carboxyaldehyde4-MethoxybenzaldehydePyridine-3-carboxyaldehydeCinnamaldehyde4-HydroxybenzaldehydeFuran-2-carboxaldehyde3-HydroxybenzaldehydeHexan- I -al2-HydroxybenzaldehydePerillaldehydeOctan- I -al

261857469311175445314590111Ill265

0.572.83.95.16.5

2016261518596372187152460

1961241741202122181417284

2301409876105

78704457367253752922503231451929

131831168014114494485615693655170

72644152336649682620462929411726

4.944.542.041.761.521.301.261.081.061.060.660.590.460.26

12623.110.310.25.073.303.082.621.821.100.790.460.390.220.110.06

highest specificity constants were obtained with aromaticaldehydes with the smallest substituent groups. Forexample, the specificity constant of benzaldehyde was byfar the highest, and then monofluorobenzaldehydes werebetter substrates than monohydroxybenzaldehydes andmonomethoxybenzaldehydes. The ability of benzaldehydedehydrogenase II to oxidize certain aldehydes probablyrelates to their hydration properties. Aromatic aldehydesare essentially unhydrated in solution (see, e.g., Klinman,1972) whereas aliphatic aldehydes exist to variousdegrees in solution as the hydrated gem-diol form(Bodley & Blair, 1971).The specificity of benzaldehyde dehydrogenase II

suggests that the active site may be a tight pocketstructure rather than the cleft postulated for benzylalcohol dehydrogenase. However, an interpretation ofthe ability of an enzyme to bind (and oxidize) a substratein relation to that substrate's structure is a complexfunction not only of steric effects but also of hydrogen-

bonding, electronic and hydrophobic effects (Klinman,1972). For example, the aromatic ring of the substratesof benzyl alcohol dehydrogenase and benzaldehydedehydrogenase II appears to confer specificity. Bindingof the ring may contribute greatly to the binding energyand therefore to the energy for catalysis.

Induction and substrate specificities in relation to growthBenzyl alcohol and benzaldehyde both induce benzyl

alcohol dehydrogenase and benzaldehyde dehydrogenaseII in A. calcoaceticus (Livingstone et al., 1972), but ingeneral the substrate specificities of both benzyl alcoholdehydrogenase and benzaldehyde dehydrogenase IIappear to be different from their induction specificities.For example, thiophen-2-methanol, pyridine-3-methanoland pyridine-4-methanol induce the enzymes (Living-stone et al., 1972) but only thiophen-2-methanol is asubstrate for benzyl alcohol dehydrogenase. Cinnamyl

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alcohol on the other hand is a substrate for benzylalcohol dehydrogenase but is not an inducer (Livingstoneet al., 1972), and pyridine-3-carboxaldehyde is both aninducer (Livingstone et al., 1972) and a substrate ofbenzaldehyde dehydrogenase II. In other bacterialsystems [e.g. the lac system of Escherichia coli (Lewin,1974) and the ami system of Pseudomonas aeruginosa(Clarke, 1984)] inducer and substrate specificities areoften very different, suggesting that in general the relevantenzymes and repressors had different evolutionary origins(Baumberg, 1981).The combined substrate and inducer specificities of

both benzyl alcohol dehydrogenase and benzaldehydedehydrogenase II determine which substrates can beutilized as carbon sources for A. calcoaceticus providedthat there is the ability to take up the compound andthat the enzymes of the ortho ring-cleavage pathwaywill accept their products. Fewson (1967) found thatA. calcoaceticus N.C.I.B. 8250 would grow on benzylalcohol, benzaldehyde, 2-hydroxybenzyl alcohol (salicylalcohol), 2-hydroxybenzaldehyde (salicylaldehyde),4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde,4-hydroxy-3-methoxybenzyl alcohol (vanillyl alcohol),4-hydroxy-3-methoxybenzaldehyde (vanillin) and 3,4-di-hydroxybenzaldehyde. No other substituted benzyl alco-hols or benzaldehydes would serve as growth substrates.As 4-hydroxy-3-methoxybenzaldehyde and 3,4-di-hydroxybenzaldehyde are not substrates for benzalde-hyde dehydrogenase II (Table 2), growth must occur as aresult of the oxidation of these compounds by anotheraldehyde dehydrogenase. Other aromatic alcohols, suchas 3-hydroxybenzyl alcohol or 3-methoxybenzyl alcohol,although oxidized via benzyl alcohol dehydrogenase andbenzaldehyde dehydrogenase II, are not utilized byA. calcoaceticus as growth substrates (Fewson, 1967).Oxygen-utilization patterns indicate that these com-pounds are not metabolized past the corresponding acid(Kennedy & Fewson, 1968), the specificity of the ring-cleavage enzymes dictating which acids will be furthermetabolized. The restricted specificity of the ring-cleavage enzymes also appears to be responsible for theinability of A. calcoaceticus to grow on cinnamyl alcohol(Fewson, 1967), which is oxidized only to a cinnamicacid (Kennedy & Fewson, 1968). Cinnamyl alcohol andconiferyl alcohol are both known intermediates oflignin biosynthesis and degradation (Crawford, 1981).Although cinnamyl alcohol or, presumably, coniferylalcohol cannot serve as sole carbon sources for A.calcoaceticus, their oxidation via benzyl alcohol dehydro-genase and benzaldehyde dehydrogenase II could givesome energy to the organism and the acids producedwould then be available for metabolism by otherorganisms. It may well be that in natural environmentsthe mixed populations of micro-organisms containindividual species that can metabolize compounds onlypartially, excreting or releasing those compounds thatare then available to be utilized by other organisms. Itis presumably by this route that A. calcoaceticus andother soil bacteria encounter aromatic alcohols, alde-hydes and acids as a result of fungal degradation oflignins (Cain, 1980).

Substrate inhibition of benzaldehyde dehydrogenase IIby benzaldehyde

Benzaldehyde dehydrogenase II was inhibited byrather modest concentrations of benzaldehyde (Fig. 1).

>. 80

CX . _

0

o o 60C

O,0 m

E 40~0

0

0 .rI EN -6

m 3

(a)

>I I I

0 20

60

, 50C)I

zn*-

4C O 400 ..~>Qa-o

"0

0.'a

20cO

10m0

40 60[Benzaldehyde] (#M)

0 0.2 0.4 0.6 0.81/[Benzaldehyde] (#Mm-1)

80 100

1.0

Fig. 1. Substrate inhibition of benzaldehyde dehydrogenase II bybenzaldehyde

(a) Plot of initial velocities against benzaldehyde concen-tration at fixed NADI concentrations of 2 mm (M) or0.5 mm (0). (b) Double-reciprocal plots of the initialvelocities against benzaldehyde concentration at fixedNADI concentrations of 2 mm (M) and 0.5 mm (0). Alldeterminations were made in duplicate and all experi-mental points are shown; where a single point is given theduplicates were identical.

The apparent Km of benzaldehyde dehydrogenase II forbenzaldehyde is 0.68 /tM (MacKintosh & Fewson, 1988),and this, together with the substrate inhibition of theenzyme by benzaldehyde above 10 /UM, suggests that theenzyme has evolved to utilize low benzaldehyde con-centrations, the enzyme being saturated at concentrationsnot toxic to the enzyme or the cell. Several otherprokaryotic and eukaryotic aldehyde dehydrogenaseshave apparent Km values for their substrates of less than1 /SM and also are substrate-inhibited. These include thePseudomonas putida NADP+-linked benzaldehyde de-hydrogenase, which has an apparent Km for benzaldehydeof 0.2 /LM and is inhibited by benzaldehyde at con-centrations above 0.1 mM (Stachow et al., 1967).

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Table 4. Esterase activity with 4-nitrophenyl acetate

The dehydrogenase and esterase activities of benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase II and yeast aldehydedehydrogenase were measured as described in the Experimental section. The values shown are averages of duplicate assays,which generally agreed within 5 %. Abbreviation: N.D., not determined.

Dehydrogenase Esterase Esterase activitypH of assay activity activity relative to

(units/mg of (unit/mg dehydrogenaseEnzyme protein) of protein) activity (%)

Benzyl alcohol dehydrogenase

Benzaldehyde dehydrogenase II

Yeast aldehyde dehydrogenase

9.58.47.49.58.47.49.58.47.4

* Less than 0.5 %0 of the rate for benzaldehyde dehydrogenase II.

178N.D.N.D.7253305.7

10.02.9

N.D.0*0*

N.D.0.650.12N.D.0.330.16

N.D.N.D.N.D.N.D.1.20.4N.D.3.35.5

Esterase activityAldehyde dehydrogenases from several sources have

esterase activity with 4-nitrophenyl acetate as substrate(e.g. Feldman & Weiner, 1972). The esterase activities ofbenzyl alcohol dehydrogenase and benzaldehyde de-hydrogenase II and of yeast aldehyde dehydrogenase asa reference were therefore determined (Table 4). Benzylalcohol dehydrogenase showed no detectable esteraseactivity, but benzaldehyde dehydrogenase II was activewith 4-nitrophenyl acetate. The relative esterase activityof benzaldehyde dehydrogenase II is low compared withyeast aldehyde dehydrogenase, but the dehydrogenasespecific activity of the bacterial enzyme is much higherthan that of the yeast enzyme. The esterase activity couldnot be measured above pH 9 (i.e. near to the pHoptimum for dehydrogenase activity; MacKintosh &Fewson, 1988), because of the high blank rates of non-enzymic hydrolysis. There has been much debate as towhether the dehydrogenase and esterase activities are atone or two active sites, and whether a common reactivethiol group is involved in their reaction mechanisms(Duncan, 1985). However, the argument seems to havecome down on the side of the single-active-site modelwith the two reactions having a common thioester acyl-intermediate step (Duncan, 1985; Loomes & Kitson,1986).

Effects of thiol-blocking reagents on the activities ofbenzyl alcohol dehydrogenase and benzaldehydedehydrogenase II

Each enzyme exhibited different sensitivities to thevarious reagents tested (Table 5). Benzyl alcohol de-hydrogenase was particularly sensitive to inactivation byiodoacetate, whereas benzaldehyde dehydrogenase IIwas most sensitive to 4-chloromercuribenzoate. Whenbenzyl alcohol dehydrogenase and benzaldehyde de-hydrogenase II were incubated with 4-chloromercuri-benzoate or N-ethylmaleimide in the presence of benzylalcohol, benzaldehyde or NAD+ there was evidence ofprotection against inhibition (results not shown). Thecofactor appeared to be less protective than either thealcohol or the aldehyde. Protection of benzyl alcohol

dehydrogenase and benzaldehyde dehydrogenase IIagainst inhibition by these reagents suggests (but doesnot prove) that the inhibition is active-site-directed.The inhibition of benzyl alcohol dehydrogenase

and benzaldehyde dehydrogenase II by thiol-blockingreagents is a common feature shared with all bacterialaromatic alcohol dehydrogenases and aromatic aldehydedehydrogenases that have been examined and also withthe mammalian and yeast alcohol dehydrogenases andaldehyde dehydrogenases. The reaction mechanism pro-posed for horse liver and yeast aldehyde dehydrogenasesinvolves a nucleophilic attack on the aldehyde by areactive thiol group (Jakoby 1963; Feldman & Weiner,1972), which is sensitive to modification by thiol-blockingreagents. The sensitivity of benzaldehyde dehydrogenaseII to such reagents may therefore be indicative of ithaving a similar reaction mechanism. This idea isstrengthened by the observation described above thatbenzaldehyde dehydrogenase II exhibits esterase activitywith 4-nitrophenyl acetate as substrate, as do both horseliver and yeast aldehyde dehydrogenases (Feldman &Weiner, 1972).

Benzyl alcohol dehydogenase was particularly sensitiveto inhibition by iodoacetate. Horse liver alcohol dehydro-genase is also inhibited by iodoacetate (Sund & Theorell,1963). Several bacterial aromatic alcohol dehydrogenasesare also sensitive to inhibition by iodoacetate, but not tothe same extent as benzyl alcohol dehydrogenase. Thusbenzyl alcohol dehydrogenase purified from Ps. putida T-2 was much more sensitive to 4-chloromercuribenzoatethan it was to iodoacetate (Suhara et al., 1969).Benzaldehyde dehydrogenase II and other bacterialaromatic aldehyde dehydrogenases (Ballal et al., 1967;Kiyohara et al., 1981) are also much less sensitive toinhibition by iodoacetate. Both benzyl alcohol dehydro-genase and benzaldehyde dehydrogenase II can beinhibited by low concentrations of 4-chloromercuri-benzoate. Several bacterial aromatic alcohol and alde-hyde dehydrogenases seem to be especially sensitive to 4-chloromercuribenzoate (Ballal et al., 1967, 1968; Suharaet al., 1969; Kiyohara et al., 1981), and as the reagentcontains a benzene ring structure its potency couldperhaps also be an active-site-directed phenomenon.

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Table 5. Inhibition of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II by thiol-blocking reagents

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were diluted in 100 mM-potassium phosphate buffer, pH 7.5,containing various concentrations of thiol-blocking reagents and then incubated on ice. Samples were taken for assay atintervals. The concentrations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II proteins in the incubationmixtures and in the enzyme assays were 1.44 ug/ml and 9.6 ng/ml, and 6.08 ag/ml and 40.5 ng/ml, respectively. Plots ofpercentage activity against time were drawn and the 500% inhibition points were estimated for the different inhibitorconcentrations.

Benzyl alcohol dehydrogenase Benzaldehyde dehydrogenase II

Inhibitor con- Time to reach Inhibitor con- Time to reachInhibitor centration (#M) 50 % inhibition (s) centration (uM) 50% inhibition (s)

lodoacetate

lodoacetamide

4-Chloromercuribenzoate

N-Ethylmaleimide

0.11

I010

10100200500

3030

54030013533023530

48030

100010000

10100

10002

10100

1000

117030

8606002101000270

1200220160

Effects of metal-ion chelators on the activities ofbenzyl alcohol dehydrogenase and benzaldehydedehydrogenase II

Both benzyl alcohol dehydrogenase and benzaldehydedehydrogenase II were assayed in the presence of thefollowing metal-ion chelators: 1O mM-EDTA, 1 mM-2,2'-bipyridyl, 1 mM-pyrazole and 1 mM-2-phenanthroline.The assay concentrations of benzyl alcohol and benz-aldehyde were 0.02 or 0.2 mm and 0.002 or 0.01 mMrespectively. No inhibition of benzyl alcohol dehydro-genase or benzaldehyde dehydrogenase II was observed.However, in a control experiment horse liver alcoholdehydrogenase was inhibited by 72% when assayed inthe presence of 1 mM-pyrazole. The activities of benzylalcohol dehydrogenase and benzaldehyde dehydrogenaseII in crude extracts of A. calcoaceticus were alsounaffected by metal-ion chelators.The insensitivity of benzyl alcohol dehydrogenase to

inhibition by metal-ion chelators is another property itshares with other bacterial aromatic alcohol dehydro-genases (Suhara et al., 1969; Jaeger et al., 1981). Incontrast, horse liver and yeast alcohol dehydrogenasesare both sensitive to chelating agents and this is knownto be a result of the presence of zinc atoms at the activesites (Sund & Theorell, 1963). The bacterial aromaticalcohol dehydrogenases thus appear to be members ofthe group of non-zinc dehydrogenases (Jornvall, 1986;J6rnvall et al., 1987).

Inhibition by non-substrate alcohols and aldehydesBenzyl alcohol dehydrogenase and benzaldehyde de-

hydrogenase II were assayed in the presence of certainalcohols and aldehydes that had previously been shown(Table 2) not to be substrates for the enzymes. Benzylalcohol dehydrogenase was slightly inhibited by cyclo-hexanol and hexahydrobenzyl alcohol (results notshown). However, benzaldehyde dehydrogenase II was

inhibited by several non-substrate aldehydes, and 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes wereparticularly potent inhibitors (Table 6). Substitution inthe ortho position of the ring may cause the aldehydes tobind incorrectly for oxidation but with an increasedaffinity, thus competing out the active substrates ofbenzaldehyde dehydrogenase II. The ability of the

Table 6. Inhibition of benzaldehyde dehydrogenase II by non-substrate aldehydes

Benzaldehyde dehydrogenase II was assayed in thepresence of non-substrate aldehydes and the standardconcentrations of benzaldehyde (0.01 mM). The valuesshown are averages of duplicate assays, which generallyagreed within 50%. The 1000% activity of benzaldehydedehydrogenase II was 78 units/mg of protein.

ActivityNon-substrate aldehyde present (%)

None0.01 mM-2-Bromobenzaldehyde0.1 mM-2-Bromobenzaldehyde'0.01 mM-2-Chlorobenzaldehyde0.1 mM-2-Chlorobenzaldehyde0.1 mM-2,6-Dichlorobenzaldehyde0.1 mM-3,4-Dimethoxybenzaldehyde0.01 mM-2-Fluorobenzaldehyde0.1 mM-2-Fluorobenzaldehyde0.05 mM-4-Hydroxy-3-methoxybenzaldehyde0.01 mM-4-Isopropylbenzaldehyde0.1 mM-4-Isopropylbenzaldehyde0.1 mM-2-Methoxybenzaldehyde0.1 mM-2-Methylbenzaldehyde0.1 mM-3-Methylbenzaldehyde0.1 mM-Pentafluorobenzaldehyde

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substituted aldehydes to inhibit benzaldehyde dehydro-genase II appears to be lowered as the size and numberof the substituent groups on the ring increases. It hasbeen reported that the reactivity of yeast and rat liveraldehyde dehydrogenases towards halobenzaldehydes islargely dependent on the position and size of thesubstitution (Rietveld et al., 1987). In particular, increas-ing the size of the halogen substituent in the orthoposition drastically decreased the enzymes' reactivitiesand it was suggested that it is the steric hindranceof the ortho-substituted benzaldehydes that results inthe enzymes' decreased reactivities (Rietveld et al.,1987).Those substrate analogues that are not oxidized by the

enzymes and that are not inhibitors could perhapsbe anti-inducers of benzyl alcohol dehydrogenaseand benzaldehyde dehydrogenase II expression. Anti-inducers can be used to isolate constitutive strains, andenzyme inhibitors can be used to isolate over-expressingstrains, as has previously been achieved with the enzymesspecific for mandelate metabolism in Ps. putida and A.calcoaceticus (Hegeman & Root, 1976; Fewson et al.,1978). Unfortunately, in preliminary experiments wefound the substituted benzaldehydes to be very toxic togrowth.

Possible metabolic inhibitorsThe effects of selected metabolites on the activities of

benzyl alcohol dehydrogenase and benzaldehyde de-hydrogenase II were examined. The enzymes wereassayed in the presence of 0.2 mm of the followingcompounds: L-mandelate, phenylglyoxylate, benzoate,succinate, acetyl-CoA, ATP and ADP. The assayconcentrations of benzyl alcohol and benzaldehyde were0.02 or 0.2 mm and 0.002 or 0.01 mm respectively. Noinhibition of either enzyme activity was observed withany of the compounds. This is consistent with the factthat no signs of feedback inhibition of the enzymes'activities were observed during investigations of theregulation of benzyl alcohol metabolism in intact cells ofA. calcoaceticus (Beggs et al., 1976; Beggs & Fewson,1977). This pathway is clearly not subject to regulationby feedback inhibition.

ConclusionNow that benzyl alcohol dehydrogenase and benz-

aldehyde dehydrogenase II have been partially charac-terized, we can investigate their relationships to themandelate-induced benzaldehyde dehydrogenase I fromA. calcoaceticus (Livingstone et al., 1972) and to otherbacterial dehydrogenases, including the chromosomallyand plasmid-encoded enzymes of Ps. putida (Collins &Hegeman, 1984; Williams & Murray, 1974). Comparisonof these enzymes might give some indication of how thepathways have been acquired by the organisms.

R. W. M. thanks the Science and Engineering ResearchCouncil for a research studentship.

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Received 29 January 1988/5 April 1988; accepted 13 April 1988

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