Microbial metabolism of mandelate: a microcosm of diversity

FEMS MicrobiologyReviews 54 (1988) 85-110 Published by Elsevier

85

FER 00086

Microbial metabolism of mandelate: a microcosm of diversity Charles A. F e w s o n Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.

Received 5 May 1987 Accepted 5 August 1987 Key words: Aromatic; Bacterium; Enzyme; Evolution; Fungus; Genetics

1. S U M M A R Y This review highlights the diversity of prokaryotic and eukaryotic microorganisms that can metabolise mandelate and it describes how a wide range of compounds related to mandelate is formed in many environments. The chief aspects that are summarised include the various pathways whereby mandelate and its structural analogues are converted into catechol or protocatechuate, the properties of the enzymes that are involved in the pathways, and the regulation and genetics of the pathways. The review incorporates the idea that the study of peripheral metabolic pathways is particularly useful for illuminating evolutionary speculations and it concludes with a list of questions that need to be answered.

2. I N T R O D U C T I O N Supniewski [1] discovered that the 'Bacille pyocyanique' converted mandelate into benzoate. However, it was the wide-ranging work of Stanier and his colleagues [2-8] which elucidated the mechanism of mandelate degradation in some pseudomonads and introduced the mandelate Correspondence to: C.A. Fewson, Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.

pathway as a worthy topic of biochemical, genetic and physiological investigation and so of evolutionary speculation. Subsequent work has estabfished this pathway as a paradigm of microbial metabolism. Mandelate, which is by no stretch of the imagination a common substance, is degraded by at least as many different pathways as is glucose - perhaps by more. Some of these pathways may have had independent origins, presumably by the recruitment of suitable enzymes from other areas of metabolism. Even in those organisms in which the enzymes are very similar and probably had common origins, their regulation is often quite different. This is not surprising in view of the fact that the ability to metabofise mandelate, although not so widespread as to be unremarkable, is found in a varied selection of Gram-negative and Grampositive bacteria as well as in filamentous fungi and yeast. Many of the enzymes are of interest for their own sakes: some have unusual and intriguing mechanisms, a few are membrane-bound, several involve the incorporation of molecular O2, and some seem to exhibit crypticity.

3. M A N D E L A T E A N D P O U N D S IN N A T U R E

RELATED

COM-

Mandefic acid (2-hydroxy-2-phenylacetic acid, a-hydroxybenzeneacetic acid, a-hydroxyphenyl-

0168-6445/88/$09.10 © 1988 Federation of European MicrobiologicalSocieties

86

acetic acid, phenylglycollic acid) exists in two enantiomeric forms (Fig. 1). The pK a is 3.37 so under most physiological conditions the predominant form will be the carboxylate mandelate ion. There are few reports of the occurrence of free mandelate in Nature. It has been found in tissues of plants such as wheat leaves [9] and grapes [10]. Many plants accumulate glycosides of mandelonitrile, e.g. prunasin and sambunigrin are fl-glucosides of D- and L-mandelonitrile respectively, and amygdalin, which is formed in almonds and in peach and apricot pits, is the fl-gentiobioside of D-mandelonitrile [11]. However, these compounds are formed in plants from L-phenylalanine via mandelonitrile rather than from mandelate itself [12]. Further, their breakdown by plant glucosidases gives mandelonitrile which in turn is hydrolysed by mandelonitrile lyase to give HCN and benzaldehyde [11,13,14] (Fig. 2a). The toxicity of these compounds results in the notorious infertility of soil under peach trees [13,15]. A variety of arthropods (Lepidoptera, Coleoptera, Chilopoda, Diplopoda) produce repellant mixtures of HCN and benzaldehyde from D-mandelonitrile or its glycosides such as prunasin which they are able to store in substantial amounts [16,17]. If any of these mandelonitriles find their way into soil or other environments then presumably they may give rise to free mandelate by hydrolytic reactions (Fig. 2b, c). Mandelate and phenylglyoxylate (benzoylformate) are continually added to natural environments from animal urine. Normal human urine concentrations of mandelate are 1-15 #M and in phenylketonuria as much as 600 /LM has been reported [21]. Mandelate is formed by the breakdown of naturally-occurring compounds [22] and is also found after industrial or domestic exposure to styrene [e.g. 23-25] (Fig. 3), doping

COOH H.--C--'OH

© (R)-form D(-)-mandellc acid

COOH HO~C

-" H

© (S)-form k(+)-mandelic acid

Fig. 1. Enantiomers of mandelic acid.

CN

CONH 2

I

©

l

H-C-OH

c mandelamlde

mandelonitrile

COOHA H ~

2°

I CHO

benzaldehyde

H -- C-- OH

mandel ic acid

Fig. 2. Possible pathways for the enzymic breakdown of mandelonitrile by (a) mandelonitrile lyase (E.C. 4.1.2.10), (b) nitrilase (nitrile aminohydrolase, E.C. 3.5.5.1) or (c) successive hydrations by nitrile hydratase and an amidase [see 14,18-20].

with the stimulant pemoline [21,26], administration of ethylbenzene or acetophenone [27] (Fig. 3), or ingestion of drugs such as N-(phenylethyl)-3,3diphenylpropylamine or 2-(3-hydroxyphenylethylamino)pyridine [21]. Mandelic acid is also a component of various pharmaceutical formulations, e.g. mandelyltropeine, a mydriatic agent [28], 3,3,5-trimethylcyclohexanyl mandelate, a vasoactive substance [29], and methenamine mandelate, a urinary antiseptic [e.g. 30,31], and their administration presumably leads to the excretion of fairly large amounts of mandelate. A dilute solution of mandelic acid is used in some hospitals as a bladder irrigation fluid to prevent urinary tract infections associated with urethral catheterization [32]. Mandelate is also incorporated in some selective media used for the isolation of bacteria such as Salmonella spp. [33,34] and mandelate esters have been used as repellants for Tribolium confusum [35]. It is therefore possible that substantial amounts of mandelate are continually entering many environments even though there seem to be no reports of its concentration in soils, natural waters, etc.; this could mean that it has not been systematically looked for or that it is rapidly metabolised. The facile isolation of microorganisms able to metabolise mandelate has been contrasted with the lack of evidence for significant amounts of

87

ICH CH

CH

/©....

ethylbenzene

styrene

/-CH2

CH3

H2C,~ H~/0

© L-styreneoxide

d

acetophenone

D-styrene oxlde

~H20H

elH2OH H--C--OH

ClH2OH C~O

©

©

hydroxyacetophenone

D-phenyl 1,2-ethanedlol

k-phenyl 1,2-ethanediol

/ f OOH

COOH I H--C--OH

/

/

©

L-mandelic acid

D-mandel ic acid

COOH / I

© C~O

phenylglyoxyllc acld Fig. 3. Mammalian metabolism of styrene, ethylbenzene and acetophenone to form urinary mandelate and phenylglyoxylate [25,27].

mandelate in nature. This apparent paradox, coupled with the recognition that many of the mandelate enzymes tolerate ring-substitution and are active with hydroxylated and methoxylated substrates, has led to an assumption that hydroxyand methoxy-substituted mandelates derived from lignin and related natural products are the prin-

cipal substrates of the organisms in nature [36,37]. Unfortunately, there appears to be no good evidence for this. The phenylpropanoid monomeric units of lignin might be expected to yield C6-C 3 or C6-C 1 (as a result of fl-oxidation) products after depolymerisation, not C6-C 2 compounds such as mandelate. This turns out to be the case

88

mandelate) and 3,4-dihydroxymandelate, together with related compounds such as 4-hydroxy-3methoxyphenylglycol and 3,4-dihydroxyphenylglycol are metabolites of adrenaline (epinephrine) and noradrenaline (norepinephrine) and substantial amounts are continually being excreted into the environment in the urine and faeces of normal animals, and even greater amounts are excreted in certain pathological conditions (see Fig. 4 for structures of these compounds) [41-44]. Octopamine, an invertebrate neurotransmitter also found in mammalian brain, is metabolised to 4-hydroxy-

because the commonest low-M r aromatic compounds in soil seem to be C6-C 3 (e.g. p-coumarate and ferulate) and C6-C ] (e.g. protocatechuate and vanillate, vanillin and 4-hydroxybenzaldehyde) [38,39] and even these are present in the soil water at concentrations of only about 10 I~M [40], although of course pool size gives no indication of flux. Nevertheless, whilst ring-substituted mandelates may not be formed from lignin, there is a great deal of evidence that they are commonly formed from other precursors. D ( - ) - 4 - H y d r o x y 3-methoxymandelate (vaniUylmandelate, vanil-

+~H2 - CH3

+~H3 ~H2

CH +NH3

~H2

H--C--OH

H--C--OH

noradrena] ine

COOH

OH

octopamlne

COOH I H--C--OH

COOH I

I

H--C--OH

@OH

H~OCH3 OH D(-)-4-hydroxy-3-methoxYmandeitc acid

0

OH

OH D(-)-3,4-dlhydroxymandelic acid

o(-)-4-hydroxymandellc add

~

~H20H

H20H H-- C-- OH

H--C--OH ~OCH OH

0

OH adrenaline

OH

3 OH

3,4-dlhydroxyphenyl

4-hydroxy-3-methoxyphenylglycol

glycol

CH20H

~H--NH--CO--CHCI2 H--C--OH

NO2 chlorampnenlcol

COOH I H--C--OH

0

NO2

4-nltromandelic acid

Fig. 4. Adrenaline, octopamine, chloramphenicol and structural analogues of mandclic acid.

89

COOH I H2N--~-H

COOH I

H--C--NH2

6 I

COOH ~H'NH2I

6

d

L-phenylalanlne

D-Phenylalanlne

-....

OH

L-tyrosine

COOH I C~O

COOH I C=O I

I

6

OH

phenylpyruvic acid

4-hydroxyphenylpyruvtc

acid

CHO

CHO I

6

OH

phenylacetaldehyde

4-hydroxyphenylacetaldehyde

C00H

COOH I

6

I

.

¢

phenylacet ic acid

OH 4-hydroxyphenylacetlc

acid

~OOH

COOH I

6

d

mandeltc acid

OH

4-hydroxymandellc acid

"""...,.°.. "...

•~

~..

,..-'"

...'" .

further metabolism

Fig. 5. Tentative pathways for the formation of mandelate and 4-hydroxymandelate by the fungus Aspergillus niger (..*), the phytoplankton Isochrysis galbana and Navicula incerta ( -~ ) a n d the red alga Odonthalia floccosa (---*) [based on 48-50].

90

mandelate which is excreted [45]. L-Phenylalanine, phenylacetate and L-tyrosine are converted into mandelate and 4-hydroxymandelate by a wide range of organisms including Polyporus hispidus [46], Penicillium chrysogenum [47], Aspergillus niger [48], phytoplankton [49] and red algae [50], although some of the enzymes thought to be involved in the postulated metabolic pathways (Fig. 5) have not been identified and in particular the mechanism whereby the a-carbon of phenylacetate (or 4-hydroxyphenylacetate) is hydroxylated to give mandelate (or 4-hydroxymandelate) is not known. Crowden [51] has suggested on the basis of isotope incorporation experiments that the fungus Polyporus tumulosus forms 4-hydroxymandelate, 2,5-dihydroxymandelate and 3,4-dihy-

droxymandelate from shikimate. Similarly, 3carboxymandelate and 3-carboxy-4-hydroxymandelate are formed in various plants [52]. Lastly, Lingens and colleagues [53] have proposed that a pathway for the microbial degradation of chloramphenicol gives rise to 4-nitromandelate.

OF MICROORGANISMS THAT CAN UTILISE MANDELATE 4. O C C U R R E N C E

Strzelczyk et al. [54] found that upto about a quarter of all the benthic microorganisms isolated from the sandy sediment and 'dy'-type sediment of a eutrophic lake could grow on mandelate and the mandelate-utilisers included members of the

Table 1 Microorganisms that are able to utilise mandelate Species

(i) Bacteria A cinetobacter calcoaceticus Arthrobacter spp. Arthrobacter-Corynebacterium b Azotobacter beijerinckii c Bacillus sphaericus ¢ Bacillus spp. b Nocardia spp. b Pseudomonas aeruginosa Pseudomonas caryophylli Pseudomonas convexa e ( putida ? [58]) Pseudomonas fluorescens Pseudomonas multivorans ( cepacia ) Pseudomonas putida (ii) Fungi Aspergillus flavus ¢ Aspergillus niger d Byssochlamys fulva c Neurospora crassa c,d Rhodotorula graminis c Unidentified yeast a b c d

Enantiomer used a

Reference

3/106: D or L; 1/106: L only; 5/106: phenylglyoxylate 8/35: D or L; 1/35: D only 1/130: unspecified enantiomer; and 3/150 phenylglyoxylate

[60] [61] [62]

D and L L only

All 29 strains tested used L but not D (1 strain did not grow on phenylglyoxylate) Some strains use L

[54] [58] [58] [541 [54] [571

3/94: L only; 21/94: phenylglyoxylate 4/17: L only; 15/17: phenylglyoxylate 5/41: D only; 3/41: D or L; 16/41: phenylglyoxylate

[62a] [63] [57] [57] [57]

D or I~ or D or D or D or

[64] [651 [66] [671 [681

D and L

L

L L L L L

[58]

Incidence of positive strains out of the total number of strains tested. Enantiomer not specified. Only one strain recorded. These strains oxidised mandelate after growth in its presence together with other carbon sources but there is no evidence as to whether they can grow on mandelate as sole source of carbon and energy.

91

Arthrobacter-Corynebacterium group, Nocardia spp., Bacillus spp. and Gram-negative rods. Laboratory-grown activated sludge and treatment plant activated sludge contained large numbers of mandelate-utilising organisms [55,56]. A wide range of both prokaryotic and eukaryotic microorganisms can metabolise one or both enantiomers of mandelate (Table 1). Many of these observations were made with organisms that were already held in culture collections. However, standard enrichment procedures using mandelate as the sole source of carbon allow the isolation of mandelate-utilising organisms [e.g. 37,57,58]. Alternate transfer between media containing the two separate enantiomers gives organisms able to use both D- and L-mandelate and these usually have two stereospecific mandelate dehydrogenases although in a few cases (so far always Pseudomonas putida biotype A) there is a mandelate racemase [37]. Organisms with only a single mandelate dehydrogenase and no racemase can be isolated by replica-plating from agar containing one enantiomer onto medium containing the other enantiomer and looking for colonies that fail to grow on the replica plates [57]. There is no evidence about the extent to which mandelate may be co-metabolised or partially metabolised by individual species of microorganisms or about the possibility of it being degraded by mixed populations. Further, all the studies have concentrated on aerobic conditions and there appears to be no report of anaerobic mandelate metabolism although many other aromatic compounds can be degraded anaerobically [e.g. 59].

5. CHEMOTAXIS TOWARDS MANDELATE Chemotaxis towards potential nutrients is presumably of selective advantage to motile microorganisms. D,L-Mandelate was found to be an effective chemoattractant for Pseudomonas putida in capillary assays of chemotaxis [69]. However, experiments with blocked mutants indicated that the bacteria were actually responding to phenylglyoxylate, the first product of mandelate metabolism in this organism. Harwood et al. suggested

that the synthesis of the putative chemoreceptor for phenylgiyoxylate could not be subject to exactly the same regulatory control as the mandelate enzymes themselves because the two processes had slightly different inducer specificities. They also concluded that the fact that cells respond to endogenously generated phenylglyoxylate suggests that the chemotactic response may be mediated by recognition at a receptor site within the cell; alternatively, the phenylgiyoxylate may be excreted and then recognised by an external receptor [69].

6. UPTAKE OF MANDELATE Mandelate is a mild antiseptic agent [e.g. 32], presumably by virtue of its ability to destroy the integrity of bacterial membranes, and it therefore supports microbial growth only at relatively low concentrations (approx. 5-20 mM is generally used in growth media). At first sight it would appear that mandelate should enter bacteria by free diffusion. Harwood and Gibson pointed out that 'Weak acids and bases with low polar characteristics can cross biological membranes with relative ease, distributing themselves so that the population of uncharged molecules is equal on both sides, and the total concentration of charged and uncharged molecular species on each side of the membrane is a function of the pH gradient' [70]. In experiments whose results were consistent with this notion, Rhodopseudomonas palustris did not appear to concentrate free benzoate against a concentration gradient; even so, the conversion of internal benzoate into benzoylCoA was so rapid that benzoate uptake was extremely effective (apparent K m < 1 #M) [70]. Nevertheless there is evidence that Pseudomonas putida possesses an inducible specific active transport system that can accumulate benzoate against a 150-fold concentration gradient, appears to be activated by the membrane potential rather than ATP hydrolysis, and has a high affinity for its substrate (Km approx. 20 gM) [71,721. Hegeman found no evidence for the existence of a mandelate permease in P. putida and even in non-induced bacteria there was rapid equilibration of mandelate across the membrane [73]. In Acin-

92

etobacter calcoaceticus, which appeared to have no permeability barrier against benzoate, there did appear to be a barrier against the entry of mandelate unless the bacteria had been grown under conditions where the mandelate-metabolising enzymes were induced [74]. Higgins and Mandelstam obtained several lines of evidence for the existence of an inducible, specific uptake system for mandelate in P. putida; uptake was sensitive to 2,4-dinltrophenol, occurred against a concentration gradient, but accumulated mandelate only if the extracellular concentration was low [75,76]. Interpretation of results of experiments on mandelate uptake may be confused by the fact that bacterial mandelate dehydrogenases are membrane bound (see Section 8.2) and so mandelate may need only to reach the periplasm in order to be dehydrogenated. Further, bacterial transport systems seem invariably to be stereospecific so this factor would have to be taken into account in testing for the existence and operation of uptake systems for D- a n d / o r L-mandelate. •

7. PATHWAYS F O R T H E M E T A B O L I S M OF MANDELATE

7.1. Pathways in prokaryotes and eukaryotes The initial attack on mandelate can be by racemization, ring hydroxylation or stereospecific dehydrogenation (Fig. 6a, b', b", e). Pseudomonas putida A.3.12 (PRS 1; ATCC 12633, NCIB 9494; previously referred to as Pseudomonas fluorescens; strain 90 of [57]) can grow on both D- and L-mandelate. D-Mandelate is converted into benzaldehyde by the successive actions of mandelate racemase, I.-mandelate dehydrogenase and phenylglyoxylate decarboxylase (Fig. 6a, b', c) and there are then two isofunctional benzaldehyde dehydrogenases, one specific for N A D and one for N A D P (Fig. 6d), which form benzoate [2-8,73,77,78]. Other strains of P. putida carry out the same reactions [37]. In addition some strains (e.g. ATCC 17426; strain 49 of [57]) can grow on D-mandelate but not L-mandelate and in this case appear to possess a D-mandelate dehydrogenase (Fig. 6b") but lack a mandelate racemase [37,58]. Finally, one strain has a mandelate

racemase and a D-mandelate dehydrogenase and so again can grow on both enantiomers of mandelate [37]. Benzoate is converted into catechol by the action of benzoate oxygenase and 3,5cyclohexadiene-l,2-diol-l-carboxylate dehydrogenase (Fig. 6i, j) [79-81]. Catechol is subject to ortho (intradiol) cleavage by catechol 1,2-dioxygenase and enters the fl-ketoadipate pathway to produce succinate and acetyl-CoA which join the amphibolic pathways and allow growth [82-851. Pseudomonas aeruginosa can grow on only L-mandelate, which is oxidised by an L-mandelate dehydrogenase and the rest of the pathway is the same as that in P. putida (Fig. 6) [86]. There is some evidence for the existence of two NADP-linked benzaldehyde dehydrogenases, although the results based on differential induction and mutational analysis were not entirely conclusive [86]. Some strains of Acinetobacter calcoaceticus (e.g. EBF 65/174) can grow on both enantiomers of mandelate and have two stereospecific mandelate dehydrogenases; some(e.g. NCIB 8250) have only an L-mandelate dehydrogenase and so can grow on L-mandelate but not D-mandelate; and some (e.g. EBF 65/65) have a D-mandelate dehydrogenase but no L-mandelate dehydrogenase [60,61, 87]. Those strains that have only a single dehydrogenase can give rise to mutants that have a second dehydrogenase, specific for the other enantiomer, and in every case the novel enzymes (which may arise by the expression of cryptic genes) are coordinately regulated along with the pre-existing enzymes [87-89]. Phenylglyoxylate is then metabolised exactly as in P. putida (Fig. 6) except that there is only a single benzaldehyde dehydrogenase [90-94]. "Pseudomonas convexa' (probably a strain of P. putida [58]) can grow on both enantiomers of mandelate but the catabolic pathway involved shows some variations on the general theme found in most other bacteria. In this case there is a racemase but this is followed by ring hydroxylation (Fig. 6e) rather than dehydrogenation; the product, 4-hydroxymandelate, is oxidatively decarboxylated directly to 4-hydroxybenzaldehyde and 4-hydroxyphenylglyoxylate is not an intermediate. The substrate for ring cleavage is proto-

93

COOH

COOH

©

H--C-OH

COOH

I

HO--C--H

OOH

COOH

I

I

HO--C-H

'

©

L(+)-mandellc acid

D(-)-mandellc acid

L-4-hydroxymandelJc acid

!

C=O

I

C----O

0 1' ©

OH 4-tlydroxyphenyl glyoxyI IC acid

Phenylglyoxyl lc acid

Ic

CHO

OH 4-hydroxybenza ldehyde

benzaldehyde

ff COOH

©

COOH

OH 4-hydroxybenzolc acid - ~

°

benzoic acid

11'

OH protocatechulc acid

H0~H 3, 5-cyclohexadlene1,2-dlol-l-carboxyl lc acid

f ring cleavage and further metabolism to succlnate + acetylCoA

11' ~ .................................~OH catechol

Fig. 6. Metabolism of mandelate in Acinetobacter calcoaceticus (-I~). Pseudomonas putida ( ~ ), P. conoexa ( . . . . >), Aspergillus niger, Neurospora crassa and Rhodotorula graminis (---~). Enzymes: a, mandelate raeemase; b t, L(+)-mandelate dehydrogenase; b", D ( - ) - m a n d e l a t e dehydrogenase; e, phenylglyoxylate decarboxylase; d, benzaldehyde dehydrogenase (there axe several isofunctional enzymes); e, t-mandelate 4-hydroxylase; f, L-4-hydroxymandelate oxidase; g, benzoate 4-hydroxylase; h, 4-hydroxybenzoate 3-hydroxylase; i, benzoate 1,2-oxygenase; j, 3,5-cyclohexadiene-l,2-diol-l-carboxylate dehydrogenase; i + j = 'benzoate oxidase'.

94 catechuate, rather than catechol as in most bacteria (Fig. 6) [63,95-97]. The filamentous fungi .4spergillus niger [65] and Neurospora crassa [67] and the yeast Rhodotorula graminis [68,98] can metabolise both enantiomers of mandelate. There are two stereospecific dehydrogenases to form phenylglyoxylate and this is then converted into benzoate. A. niger, like P. putida, has an NAD-linked as well as an NADPlinked benzaldehyde dehydrogenase [65]. The subsequent pathway differs from that found in P. putida or Acinetobacter calcoaceticus in that benzoate undergoes two successive ring hydroxylations to form protocatechuate as the substrate for ring cleavage (Fig. 6). A similar pathway seems to operate in Aspergillus flavus [64] and Byssochlarnys fulva [66,99]. The metabolism of the products of ring cleavage has been extensively dealt with elsewhere [e.g. 83,84,100,101] and is beyond the scope of the present review. The properties of the fl-ketoadipate pathway enzymes, their regulation and possible evolutionary relationships, have been the subject of a fascinating series of papers by Stanier, Ornston and their colleagues [e.g. 100,102,103] and are of considerable significance to the more general question of evolution of metabolic pathways.

7.2. Degradation of a range of compounds oia the mandelate pathway A wide variety of organic substrates can be converted into amphibolic intermediates by being channelled into the mandelate pathway (Fig. 7) and this may be part of the reason why the mandelate pathway is so common in microorganisms.

7.3. Degradation of ring-substituted mandelates Many of the mandelate enzymes can tolerate extensive ring-substitution, especially at the 3-, 4and 5-positions. Several organisms can therefore metabolise, or partially metabolise, an extensive array of mandelate analogues (Fig. 8). Pseudomonas putida [115], Acinetobacter calcoaceticus [90,91] and Aspergillus niger [65,116] can convert one or both enantiomers of 4-hydroxymandelate into protocatechuate. A. calcoaceticus also forms

protocatechuate from L-3,4-dihydroxymandelate and L-4-hydroxy-3-methoxymandelate [90,91]. P. putida can oxidise 4-hydroxy-3-methoxymandelate [117-119] but the enantiomer that is metabolised depends on whether the strain possesses an Lmandelate dehydrogenase or a D-mandelate dehydrogenase because mandelate racemase is unusually specific amongst the mandelate enzymes and cannot use 4-hydroxy-3-methoxymandelate (or 3,4-dihydroxymandelate) as substrate [58,120]. This lack of substrate ambiguity invalidated claims to be able to use an enzyme preparation from P. fluorescens strain A.3.12 to measure normal urinary 4-hydroxy-3-methoxymandelate [121], which is the D-enantiomer [120], although use of an appropriate strain (P. putida MB-15, ATCC 17426) does give an acceptable procedure [119]. In practice urinary 4-hydroxy-3-methoxymandelate concentrations are usually measured by chemical procedures such as mass fragmentography [122], high-performance liquid chromatography [e.g. 123] or gas chromatography [e.g. 124]. The much more restricted substrate specificity of the ring-cleavage enzymes and of the enzymes of the fl-ketoadipate pathway means that compounds can only be completely oxidised if they can be converted into an appropriate substrate for one of the oxygenative ring-cleavage enzymes, such as ¢atechol or protocatechuate [e.g. 85,101]. The result is that many substrates of the mandelate enzymes, such as 3-hydroxymandelate [90,91] (Fig. 8) or halogenated mandelates [77,91], can be only. partially metabolised; this sort of effect may nevertheless be very important in natural environments where the mixed populations of organisms may totally oxidise compounds which are beyond the metabolic scope of any individual organism. Sze and Dagley [124a] recently isolated a strain of Acinetobacter that can grow on O,L-4-hydroxy3-methoxymandelate or D,L-4-hydroxymandelate but cannot grow on either L- or D-mandelate. The organism was obtained by elective culture from a cattle yard that was presumably a rich source of urinary 4-hydroxy-3-methoxymandelate. Results of experiments with bacterial extracts indicated the presence of fairly unspecific L- and D-mandelate dehydrogenases, phenylglyoxylate decarboxylase and benzaldehyde dehydrogenase. The prod-

95 CO,NH2 I HO--C--H

CHO

co, I OCH3

C~O

H ~ mandelamide methylmandelic acid

ohenylglyoxal

...... phenylacetic acid

.C

CO,OCH2CH 3 I

.lb." ~ ~ ~ mandellc acid

COON

1..41...__ 6

phenyl 1,2-ethanediol

~

H--C--NH2

H0--~--H~.~ ~1~

styrene

ICOOH

D-phenylglycine

k Phenylglyoxyllc acid

6 v

6

L-phenyIgiycine be

COOH

mandelonitrile

© benzoic

acid

Fig. 7. Degradation of several precursors via the mandelate pathway. Examples of the various conversions are: a and b, Pseudomonas putida [104] and Flavobacterium sp. [105]; c, Penicillium chrysogenum [47], mechanism uncertain, see Section 3; d, Pseudomonas putida [77], Lactobacillus sp. [106], Bacillus sp. [107]; e, Pseudomonas putida [77,108]; f and g, Pseudomonas putida [77]; h, Pseudomonas putida [77]; i, Acinetobacter calcoaceticus [90-93,109,110], Pseudomonas putida [3,111]; j, see Fig. 2 and Section 3; k, Pseudomonas putida containing TOL plasrnid [e.g. 112]; 1, occurs in mammalian tissues (see Fig. 3) but not so far found in microorganisms [see e.g. 113,114].

ucts of 4-hydroxy-3-methoxymandelate and 4-hydroxymandelate metabolism were shown to be 4-hydroxy-3-methoxybenzoate and 4-hydroxybenzoate, respectively, and these were converted into 3-O-methylgallate and protocatechuate by two NADPH-dependent hydroxylases. There appeared to be no benzoate oxygenase activity, which would

explain the failure of the organism to grow on mandelate. Surprisingly, 3-O-methylgallate and protocatechuate were shown to be subject to meta (extradiol) ring cleavage. This appears to be the first report of mandelate metabolism in which ortho fission of the benzene nucleus is not involved.

96

COOH I CHOH

COOH I CHOH

0

~OH

mandellc acid

~OH

2-hydroxymandeltc acid

COOH I

COOH I CHOH

~OOH CHOH

OH 4-hydroxy-

3-hydroxymande]lc acid

COOH I

mandellc acid

COOH I OH

CHO

CHO

COOH I

~OH "-~f~ OCH3 OH OH 3,4-dlhydroxy4-hydroxy-3-methoxv-

mande]lc acid

mandellc acid

COOH

COOH

COOH

I

!

I

OH

~OH OH

OH

H~

CHO

~OH

COOH I CHOH

H~

~I~OH OH

COOH

OCH3

COOH

COOH

OH

OH

COOH

COOH

OH

OH

OCH3

OH

OH

'~

OH

"OCH3

protocatechutc acid "",-.

catechol

.....

..,,.,

.....

,°..,

.... ~

.,all- " "

ringcleavage and further metabolism to succinate + acetylCoA Fig. 8. Metabo~smof fing-substitutedmandelates

8. PROPERTIES OF T H E E N Z Y M E S OF T H E MANDELATE PATHWAY

8.1. Mandelate racemase (Fig. 6a; E.C.5.1.2.2) This is one of the best studied of all the mandelate enzymes [58,120,125-132] and has been thoroughly reviewed [37]. The enzyme, which is found only in strains of Pseudomonas putida biotype A [37], is tetrameric, contains four identical sub-units (M, 69500), and has an absolute requirement for a divalent metal ion (e.g. Mg 2÷ or Mn 2 +) for activity. Mandelate racemase has some

byAcinetobactercalcoaceticus. Based on [91].

claims to catalyse one of the simplest of all enzymic reactions: a non-redox racemization in which no organic cofactor is involved. Various lines of evidence suggest that the reaction mechanism involves a carbanion intermediate (Fig. 9a): (a) there is a primary deuterium isotope effect of about 5 o n Vmax/Km, (b) mandelate analogues with electron-withdrawing substituents at the para position have greater Vm~x values than mandelate itself, and (c) there is a deuterium or tritium exchange between the solvent and the hydrogen on the a-carbon [37]. Initial velocity measure-

97 (a)

~oo o

i ® coo

H~C--OH

e :C--OH

+H+ ~

foo® HO~C--H

~

_H+

©

(I3) COOH

COOH

c

(S)-o~-phenylglycldic acid (R)-ct-phenylglycldlc acid Fig. 9. Mandelate racemase: (a) mechanism of action involving a carbanion intermediate [37], (b) enantiomers of the affinity label a-phenylglycidic acid [133].

ments, studies with competitive inhibitors, deuterium isotope effects and pH studies all suggest that the enzyme's active site, whilst inherently asymmetric, binds and processes the two enantiomers with remarkable symmetry [132]. Irreversible inhibition studies with the two enantiomers of the affinity label a-phenylglycidic acid [133] (Fig. 9b) revealed a 'functional asymmetry' at the active site [132]. It would appear that the enzyme has a one-base, rather than a two-base, acceptor mechanism [37,132] and results of experiments with aphenylglycidic acid have been interpreted to infer that a carboxylate group of either glutamate or aspartate attacks the epoxide ring of ct-phenylglycidic acid [127].

8.2. Mandelate dehydrogenases (Fig. 6b', b") Both L-mandelate dehydrogenase [6,7,77,134] and o-mandelate dehydrogenase [58,119] from Pseudomonas putida are membrane-bound. The equivalent enzymes from Acinetobacter calcoaceticus have been located in the inner, cytoplasmic membrane and can be released in active forms by the judicious use of ionic or non-ionic detergents [135-137]. These NAD(P)-independent dehydrogenases are usually assayed with 2,6-dichloroindophenol as electron acceptor [77,135] but the native cofactors are probably flavins [136,137] which presumably then form part of the electron transfer system in the membrane and use 02 as

the terminal acceptor. Both enzymes from A. calcoaceticus have been purified to homogeneity and have been partially characterised [136,137]. This organism also possesses a pair of NAD(P)-independent, membrane-bound lactate dehydrogenases and these have also been purified and characterised [136,138]. Comparison of the properties of the four dehydrogenases (Table 2) has led to the following conclusions: (a) The two enzymes specific for the D-enantiomers of lactate or of mandelate strongly resemble each other, as do the two enzymes specific for the L-enantiomers (ease of extraction by detergents, subunit Mr, pH optima, p l values, susceptibility to thiol reagents, and presence of F M N or FAD as cofactors) and so each of these pairs of enzymes may have had a common evolutionary origin. If so, the ability of D-mandelate dehydrogenase to dehydrogenate Dlactate (Table 2) may be an evolutionary relic of the recruitment of a lactate dehydrogenase (perhaps following gene duplication) to oxidise mandelate; or it may simply be a consequence of the similarity in chemical structures. (b) The two pairs of enzymes, although at first sight similar to each other in an overall way, are sufficiently different (especially flavin specificity and subunit Mr) that a common origin for all four enzymes is either unlikely or was much more distant. Only sequencing studies will provide the data to test these ideas. The properties of mandelate dehydrogenases from eukaryotic microorganisms are much less well understood, although it would be extremely interesting to be able to compare them with the equivalent prokaryotic enzymes. Information derived from preliminary investigations with Aspergillus niger is confusing. In strain UBC 814 there was reported to be an L-mandelate dehydrogenase that was 'soluble', reduced 2,6-dichloroindophenol and was stimulated by FAD or FMN. This strain also contained a D-mandelate oxidase that was 'particulate' (although it is not clear what organelle was involved), unstable, showed no requirement for any of the cofactors tested, and could not reduce 2,6-dichloroindophenol [65,139]. In a different strain of A. niger, isolated in Bangalore, there were D- and L-mandelate dehydrogenases, each associated with both the 'soluble' and 'par-

98 Table 2 Comparison of the lactate and mandelate dehydrogenases from Acinetobactercalcoaceticus a All the enzymes are located in the inner membrane, are NAD(P)-independent and stereospecific.

Solubilisation by non-ionic detergents (% activity released by Triton X-100 or Lubrol) Solubilisation by ionic detergents (% activity released by cholate or deoxyeholate) Subunit M r p l value pH optimum K m value (#M) for substrate Assay conditions

Inhibitors Inhibition by p-chloromercuribenzoate Cofactor (non-covalently bound)

L-Mandelate dehydrogenase

L-Lactate dehydrogenase

D-Mandelate dehydrogenase

D-Lactate dehydrogenase

43-72

55-76

87-91

83-98

4-42 44000 4.2 7.5 186

13-32 40000 < 4.0 7.5 83

Moderate

Moderate

Very severe

Severe

FMN

FMN

FAD

FAD

78-91 63-87 60000 63000 5.5 5.8 8.0 7.7 385 (in addition 308 D-lactate is a substrate but K m = 4.8 mM) 2,6-Dichloroindophenol reduction measured in the presence of N-methylphenazonium methosulphate or N-methylphenazonium ethosulphate. All require bovine serum albumin (or Triton X-100 in the case of o-mandelate dehydrogenase and D-lactate dehydrogenase) in the reaction mixture for full activity. None is affected by a wide range of chelating agents. All are moderately susceptible to several thiol-blocking reagents. All are inhibited by oxalate.

a Based on [137] with information from [135-138].

ticulate' fractions a n d each using N A D P (or to a lesser extent N A D ) as c o f a c t o r [116]. I n the y e a s t Rhodotorula graminis there is a soluble N A D - d e p e n d e n t D - m a n d e l a t e d e h y d r o g e n a s e a n d an Lm a n d e l a t e d e h y d r o g e n a s e w h i c h r e d u c e s 2,6-dichloroindophenol and may be 'membrane'-bound [98,140,141]. T h e existence in fungi o f N A D ( P ) d e p e n d e n t , s o l u b l e m a n d e l a t e d e h y d r o g e n a s e s is a striking difference f r o m the p o s i t i o n in b a c t e r i a a n d it m a y b e that these e n z y m e s h a d quite differe n t e v o l u t i o n a r y origins. N A D ( P ) - d e p e n d e n t m a n d e l a t e d e h y d r o g e n a t i o n , u n l i k e the flavin-dep e n d e n t reaction, is easily reversible [141] a n d c o u l d e x p l a i n the p o s t u l a t e d reversible i n t e r c o n v e r s i o n of 4 - h y d r o x y m a n d e l a t e a n d 4 - h y d r o x y p h e n y l g l y o x y l a t e in Polyporus tumulosus [51].

8.3. Phenylglyoxylate decarboxylase (Fig. 6c," benzoy l f ormate carboxy-lyase," E. C. 4.1.1.7) P h e n y l g l y o x y l a t e d e c a r b o x y l a t i o n was first o b served in y e a s t [142]. T h e soluble, T P P - d e p e n d e n t e n z y m e has b e e n p u r i f i e d f r o m Pseudomonas putida [143], Pseudomonas aeruginosa [144,145] a n d Acinetobacter calcoaceticus [146]. T h e e n z y m e f r o m A. calcoaceticus is a t e t r a m e r of i d e n t i c a l s u b - u n i t s ( M r 58000) a n d in this respect resembles o t h e r m i c r o b i a l T P P - d e p e n d e n t n o n - o x i d a t i v e d e c a r b o x y l a s e s for p y r u v a t e (E.C. 4.1.1.1; [147]) a n d p h e n y l p y r u v a t e (E.C. 4.1.1.43; [146]). Phenylg l y o x y l a t e d e c a r b o x y l a s e f r o m P. putida s e e m e d to h a v e a M r of 80 000 as j u d g e d b y its b e h a v i o u r o n S e p h a d e x c o l u m n s b u t in o t h e r respects was v e r y similar to the A. calcoaceticus e n z y m e

99 [143,146] although they do not cross-react immunologicaUy [148]. The enzymes from both Aspergillus niger [65,149] and Acinetobacter calcoaceticus [150] appeared to be allosterically regulated by ATP and ADP respectively but there is no evidence that this has any physiological significance. The pH optimum for the phenylglyoxylate decarboxylase from all organisms tested is low (approx. 6) and as with the yeast pyruvate decarboxylase, dissociation of sub-units and loss of TPP occur at higher pH values [143,146,147].

8.4. Benzaldehyde dehydrogenases (E.C.1.2.1.7, 1.2.1.28) There are pairs of isofunctional benzaldehyde dehydrogenase associated with the mandelate pathway in Pseudomonas putida, Aspergillus niger and perhaps P. aeruginosa (see Section 7.1) but in no case is it clear whether the separate enzymes have different functions. In addition, many organisms can grow on benzaldehyde or benzyl alcohol (Fig. 7) and in these cases additional benzaldehyde dehydrogenases are induced. Thus in both Acinetobacter calcoaceticus [36,90,92] and P. putida [151,152] there are extra benzaldehyde dehydrogenases which are under quite separate control from the mandelate enzymes and are induced only in the presence of benzaldehyde or benzyl alcohol. In view of the plethora of aromatic aldehyde dehydrogenases that are known to exist in microorganisms [153], it is surprising that so little is known about the benzaldehyde dehydrogenases associated with the mandelate pathway. The NADP-specific enzyme from P. putida has a native M r of approx. 200000, is activated by K + ions, and is strongly inhibited by thiol reagents [154]. The NAD-specific benzaldehyde dehydrogenase I from A. calcoaceticus is a tetramer of identical sub-units ( M r 4 X 55000) and is also activated by K ÷ ions [92,155]. 8.5. L-Mandelate 4-hydroxylase (Fig. 6e; L-Mandelate 4-monooxygenase) This soluble, inducible enzyme from Pseudomonas conoexa requires tetrahydropteridine, NADPH, Fe 2+ and 02 for activity and is stereospecific for the L-enantiomer of its substrate. The native M r is about 91 000 and the pH optimum is

at 5.4 [63,95,97]. Mandelate 4-hydroxylase has not yet been found in any other organisms but it would be worth looking for in fungi where hydroxylation generally seems to occur at an earlier stage in the mandelate pathway than it does in bacteria [e.g. 48,65-68].

8. 6. t.-4-Hydroxymandelate oxidase (Fig. 6f) An inducible, membrane-bound L-4-hydroxymandelate oxidase was solubilised by butan-l-ol treatment of membranes from Pseudomonas convexa and partially purified and characterised [63,96]. The enzyme catalyses the one-step oxidative decarboxylation of its substrate to form 4-hydroxybenzaldehyde without involving the intermediate formation of 4-hydroxyphenylglyoxylate. FAD and Mn 2÷ ions are required for activity. The enzyme is very specific for its substrate and various analogues (e.g. t-mandelate, 3,4-dihydroxymandelate) are not substrates and some are inhibitors. Curiously, mushroom tyrosinase catalyses a similar oxidative decarboxylation of 3,4-dihydroxymandelate with the intermediate formation of a quinone methide intermediate [156] and so L-4-hydroxymandelate oxidase may involve a parallel mode of action (Fig. 10). 8.7. Benzoate 4-hydroxylase (Fig. 6g, Fig. 11a; benzoate 4-monooxygenase, E.C. 1.14.13.12) The benzoate 4-hydroxylases from both Aspergillus niger [157] and a soil pseudomonad [158] are soluble, inducible enzymes which catalyse the conversion of benzoate into 4-hydroxybenzoate with equimolar consumption of N A D P H and 02. Tetrahydropteridine is used as cofactor [157,158].

HO--i~'-H

2H

0

4 4-hydro×ymandel acid

0 lc

1

Jlnone methtde intermediate

CHO

OH 4-hydr oxybenza1dehyde

Fig. 10. Possible mechanism of action of t-4-hydroxymandelate oxidas¢. Based on the proposed mechanism for a similar reaction catalysed by mushroom tyrosinase [156].

100

The enzyme from Rhodotorula graminis is quite different for although it uses N A D P H as electron donor, it is particulate, pteridine-independent and m a y involve a flavin as cofactor [159].

8.9. Benzoate 1,2-oxygenase (Fig. 6i, Fig. llc," benzoate hydroxylase, E.C. 1.13.99.2) The benzoate oxygenase complex of Pseudomonas aroilla has two components: an N A D H - c y -

8.8. 4-Hydroxybenzoate 3-hydroxylase (Fig. 6h, Fig. 11b; 4-hydroxybenzoate 3-monooxygenase, E.C. 1.14.13.2)

tochrome c reductase containing one F A D and one (2Fe-2S*) cluster per M r of about 37 500 and benzoate oxygenase which contains four (2Fe-2S*) clusters per M r of about 275 000 but no haem or flavin [161-163].

Crystalline 4-hydroxybenzoate hydroxylase has been obtained from several Pseudomonas species (including P. putida strain A.3.12) and it has been extensively studied as an example of a flavin-dependent mixed function oxidase [reviewed in 160]. There is a stoichiometric requirement for N A D P H and 02. F A D is used as cofactor with enzymebound F A D H 2 serving as the direct electron donor to 02 in the oxygenation reaction [160]. COOH

COOH

NADPHH+

OH q-hydroxybenzoic acid

02t~ ~NADPHH÷

:OOH

C. OH protocatechuic acid

The NAD-dependent decarboxylation of the diol substrate to catechol is catalysed by a dehydrogenase that has been purified from Alcaligenes eutrophus. The enzyme is probably a tetramer of identical sub-units ( M r 4 × 24000) and the proposed mechanism involves dehydrogenation to an unstable fl-keto acid which decarboxylates to form catechol [81].

benzoic acid O~ +

NAD(P)HH+

9. R E G U L A T I O N PATHWAY

NAD(P)

9.1. Co-ordinate expression of the mandelate enzymes

b H2Ot~ ~,.NADP+

8.10. 3,5-Cyciohexadiene-l,2-diol-l-carboxylate dehydrogenase (Fig. 6j, Fig. l ld; dihydrodihydroxybenzoate dehydrogenase)

,00 ?

3,5-cyclohexadlene 1,2-dio1-1-carboxyl ic acid dNAD÷ C02~

NADHH+

&

catechol Fig. 11. Various oxygenative hydro×ylation reactions of the mandelate pathway: a, benzoate 4-hydroxylase; b, ~hydro×ybenzoate 3-hydroxylase; c, benzoate 1,2-oxygenase; d, 3,5cyelohexadiene-l,2-diol-1 -carboxylate dehydrogenase.

OF

THE

MANDELATE

Work on the mandelate pathway led to two concepts that were influential in the early stages of development of ideas about enzyme induction in bacteria [2-5,164,165]. The first was the principle of "simultaneous adaptation': growth on a given substrate produces cells that can oxidise not only that compound but also its catabolites. The second was 'sequential induction': the idea that each enzyme is induced by its substrate and so there will be sequential induction in a catabolic pathway as successive inducers are formed by the action of each preceding enzyme. The notion of simultaneous adaptation still has some value as one line of attack in the problem of elucidating novel catabolic pathways. In general, however, the two concepts have been submerged by the realisation that (a) clusters of enzymes (operons), rather than single enzymes, are often subject to coordinate regulation, (b) enzyme products rather than substrates m a y be inducers, (c) gratuitous

101

induction is common, and (d) problems with permeation may give false negative results [3,4, 164-166]. The mandelate enzymes appear to be inducible in all the organisms, both prokaryotic and eukaryotic, that have been examined [e.g. 77,90, 91,95,98,99,116]. However, detailed measurements of the specificity and co-ordination of control have been made only with Pseudomonas putida, P. aeruginosa and Acinetobacter calcoaceticus using approaches such as (a) estimation of the degree of correlation between differential rates of synthesis of the enzymes under various conditions of induction and repression [93], (b) characterisation of blocked mutants [73,93,167], (c) induction by phenoxyacetate or thiophenoxyacetate [(phenylthio) acetate] (Fig. 12) which are gratuitous inducers in both P. putida and A. calcoaceticus [73,93], (d) co-ordinate anti-induction by 2-phenylpropionate (Fig. 12) in A. calcoaceticus [168], (e) isolation and characterisation of constitutive mutants [78,168], and (f) properties of superinducible mutants of P. putida isolated by enrichment in the presence of 2,3,4,5,6-pentafluoromandelate (Fig. 12), an inhibitor of mandelate dehydrogenase [169]. Various ring-substituted analogues can also serve as inducers [77,90,91,93]. The patterns of induction in the three species of bacteria examined are rather different from each other with respect to both specificity and degree of coordination (Fig. 13). More detailed work at the molecular level will be necessary before any worthwhile generalisations can be made but the mandelate enzymes clearly provide another exam-

COOH I ~H2 0

phenoxyacetic acid

COOH I ~H2 S

thlophenoxyacetic acid

pie of the fact that very similar enzymes may be subject to quite different regulation in different microorganisms, suggesting that evolutionary constraints on catalytic functions and on regulation are almost certainly not the same and may well be varied, accidental and obscure [170]. The enzymes converting benzoate into catechol or into 4-hydroxybenzoate and then protocatechuate appear to be controlled independently of the enzymes of the upper part of the pathway [e.g. 77,90,98].

9.2. Repression of gene expression and its consequences for growth on mandelate Mandelate enzymes are usually subject to some sort of catabolite repression, e.g. by succinate in Pseudomonas putida [165,171] and Acinetobacter calcoaceticus [93,172] or by glucose in Byssochlamys fulva [99] and Rhodotorula graminis [98]. Growth on mixtures of substrates can therefore be diauxic [98,172]. Analysis of the sequential use of pairs of substrates by P. putida and of the extent of enzyme induction and repression that occurred in appropriate mutants under various growth conditions led to the suggestion that the mandelate pathway is subject to 'multi-sensitioe end-product repression' in which products of successive coordinately induced groups of enzymes feedback repress the preceding groups of enzymes (Fig. 14) [151,171,173]. Feedback repression also occurs in the mandelate pathway of A. calcoaceticus [172]. It is not yet clear whether these feedback mechanisms are specific, or whether they are manifestations of fairly non-specific anti-induction of the

COOH, CH.CH3

2-phenylproplonlc acid

COOH, CHOH

F 2,3,4,5,6pentafluoromandelic acid

Fig. 12. Structures of some c o m p o u n d s that affect the induction of the mandelate enzymes. See text for details. In addition, an

extremely extensive array of c o m p o u n d s h a s been tested for their effects as substrates, inhibitors, inducers a n d anti-inducers of the mandelate enzymes [77,93,168].

102

D-MANDELATE mandelate racemase L-MANDELATE D-mandelate, L-mandelate

L-rnandelale dehydrogenase

L-mandelate

or

PHENYLGLYOXYLATE

phenylglyoxylate

phenylgtyoxylate

phenylglyoxylate decarboxylase

phenylglyoxylate

BENZALDEHYDE phenylglyoxylate

benzaldehyde dehydrogenase(s)

or

J3-ketoadipate

BENZOATE Acinetobacter calcoaceticus

Pseudomonas putida

Pseudomonas aeruginosa

Fig. 13. Specificity and co-ordination of induction of the mandelate enzymes in three species Of bacteria. References are in the text.

L-MANDELATE L-mandelate dehydrogenase PHENYLGLYOXYLATE phenylglyoxylate decarboxylase BENZALDEHYDE benzaldehyde dehydrogenase(s) _ BENZOATE . . . . . . I

'benzoate oxidase' CATECHOL--- - " ="

! ! I !

! I I !

iI

!I

! I

!!

J ~..ql..i ~1.1 j II II

J'= --"~- -- "J

I

catechol 1,2-oxygenase ClS,ClS -MUCONATE

1 I

I i

SUCCINATE+ ACETYL-CoA

~. . . . .

I .I

.J

I I I I I I I I

same type as that caused by 2-phenylpropionate [168]. Inducer-exclusion might also be involved [165,171] but this would depend on whether or not there are indeed specific transport mechanisms for mandelate and related compounds (see Section 6). The repressive effects are often quasi-competitive in nature and can be masked if one compound is in great excess [165,168,171]. In A. calcoaceticus mandelate metabolism dominates benzyl alcohol metabolism even though benzyl alcohol alone supports a faster growth rate than does mandelate [36,109,172]. Batch cultures of A. calcoaceticus growing on mandelate or phenylglyoxylate show an unusual non-exponential pattern. There are transient accumulations of benzaldehyde or benzyl alcohol in the growth medium caused by the limitation of mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol [172]. P. putida usually grows exponentially on mandelate as sole

Fig. 14. Multi-sensitive end-product repression in the mandelate pathway of Pseudomonas putida. Based on [151,171].

103 carbon source [77,172] but this organism also accumulates benzyl alcohol under certain conditions [174] even though it has two benzaldehyde dehydrogenases. 9.3. Feedback inhibition does not appear to be important There appear to have been no reports of specific feedback inhibition effects on any of the mandelate enzymes [see e.g. 172]. It may be that possession of peripheral catabolic pathways for utilising less common substrates that are only occasionally present in the environment is so great a selective advantage that there has been no further selection of enzymes that are susceptible to feedback inhibition. Recruitment of enzymes to serve in peripheral pathways would possibly be prejudiced if the enzymes were already subject to feedback regulation of a kind that accidentally hindered rather than helped their new functions. Nevertheless one might have thought that there would be need to regulate flux through these pathways to ensure the balanced provision of amphibolic intermediates and to avoid the accumulation of intermediates which might have deleterious effects. 9. 4. Do the mandelate enzymes exist as a multi-enzyme complex? In vivo cross-linking studies with dimethylsuberimidate and 13C N M R experiments have been used to test if there is any physical association amongst the mandelate enzymes in P. putida [174]. Five of the mandelate enzyme activities could be detected in a cross-linked complex [174]. The in vivo cross-linking by dimethylimidates has been confirmed in P. putida and A. calcoaceticus [175] but it seems to be an open question as to whether there is a specific 'mandelate complex', whether the cross-linking is non-specific, or whether some sort of transient enzyme interactions might occur [see e.g. 176].

10. G E N E T I C S O F T H E M A N D E L A T E PATHWAY In Pseudomonas putida the genes encoding the first five enzymes of the mandelate pathway ap-

pear to be linked and may even be contiguous [169,177-180]. These genes show supra-operonic clustering with genes of functionally related pathways for the degradation of 4-hydroxybenzoate, quinate, shikimate, phenylacetate, nicotinate, histidine, tyrosine and phenylalanine [180,181]. Whilst there is also some clustering of genes for related pathways in P. aeruginosa, in this organism two of the mandelate genes are separated by a group o f genes encoding enzymes for the degradation of benzoate and catechol [182,183]. In Acinetobacter calcoaceticus the mandelate genes appear to be clustered near the auxotrophic marker phe-1 but are not all contiguous with each other [89,184]. A gene responsible for the expression of the cryptic L-mandelate dehydrogenase appears to be close to a gene required for the activity of D-mandelate dehydrogenase [89]. The enzymes of peripheral catabolic pathways are often plasmid-encoded [185] and it will be interesting to see whether this turns out to be the case for the mandelate enzymes in any organism.

11. F U T U R E W O R K Specific questions that need to be answered include: (a) Does the conversion of 4-hydroxymandelate into 2,5-dihydroxymandelate observed in Polyporas tumulosas [51] involve an intramolecular rearrangement of the type invoked for the conversion of 4-hydroxyphenylacetate into homogentisate [186,187]? (b) What is the mechanism of 4-hydroxymandelate oxidase which allows it to do a task that usually requires two successive enzymes (see Section 8.6)? (c) Why do some organisms have multiple benzaldehyde dehydrogenases associated with the mandelate pathway (see Section 8.4)? (d) Is the fact that some mandelate dehydrogenases use NAD(P) rather than flavin as cofactor simply a consequence of their independent e v o l u tionary origins, or does it have functional significance in allowing the step to be reversed (see Section 8.2)? More general questions include:

104 (a) What is the precise arrangement of the structural and regulatory genes required for expression of the mandelate enzymes in different organisms? (b) Is there a multi-enzyme mandelate complex? Does channelling of the mandelate intermediates occur? Are there transient enzyme interactions in the pathway? (c) What are the molecular mechanisms regulating gene expression? (d) Are there specific transport mechanisms for these aromatic compounds? Is there chemotaxis towards the substrates? If so, how is it elicited? (e) H o w c o m m o n are cryptic genes for mandelate dehydrogenase [87-89] and other enzymes that are potentially important in these pathways? [See 188.] H o w do such enzymes become silent in the first place, what are the mechanisms that lead to their expression, and what is their ecological and evolutionary significance? (f) What are the ecological roles of mandelateutilising organisms? H o w c o m m o n are these organisms? To what extent and how often are their substrates formed? What exactly are the substrates? Finally, it will be necessary to ask: (a) Can gene cloning and sequencing, coupled with further work on the properties of the enzyme proteins, provide sufficient clues to unravel the evolutionary relationships amongst the mandelate enzymes of the prokaryotes, of the eukaryotes, and between the enzymes of both these groups of organisms? To what extent has there been gene duplication (e.g. in the case of the benzaldehyde dehydrogenases)? H a d some analogous enzymes different origins, perhaps by recruitment from quite different metabolic pathways (e.g. the mandelate dehydrogenases)? (b) Is it correct to conclude that peripheral enzymic pathways for the degradation of relatively u n c o m m o n substrates that are only spasmodically present in the environment may serve as convenient fast-running 'clocks' for following enzyme evolution, in contrast to the enzymes of the central metabolic pathways (e.g. glycolysis [189]) which are always essential, must be carefully regulated and whose evolution is therefore subject to much tighter constraints?

12. C O N C L U S I O N S The mandelate pathway provides an attractive miniature world for studying virtually every aspect of microbial biochemistry, physiology, genetics, ecology and evolution. The range of organisms and pathways involved is sufficiently great that worthwhile questions can be asked and answered, but not so excessive that too much distracting detail is accumulated.

ACKNOWLEDGEMENTS It has been a pleasure to have access to the excellent University Library here in Glasgow and in particular I am grateful for the constructive assistance of Margaret Coutts, Alison Faichney and their colleagues. M y research has been supported by grants from the Medical Research Council and the Science and Engineering Research Council.

REFERENCES [1] Supniewski, J. (1924) Transformation des compos6s aromatiques sous l'influence du Bacille pyocyanique. C. R. Soc. Pol. Biol. 90, 1111-1112. [2] Startler, R.Y. (1947) Simultaneous adaptation: a new technique for the study of metabolic pathways. J. Bacteriol. 54, 339-348. [3] Stanier, R.Y. (1948) The oxidation of aromatic compounds by fluorescent pseudomonads. J. Bacteriol. 55, 477-494. [4] Stanier, R.Y. (1950) Problems of bacterial oxidative metabolism. Bact. Rev. 14, 179-191. [5] Startler, R.Y. (1951) Enzymatic adaptation in bacteria. Annu. Rev. Microbiol. 5, 35-56. [6] Gunsalus, I.C., Gunsalus, C.F. and Startler, R.Y. (1953) The enzymatic conversion of mandelic acid to benzoic acid. I. Gross fractionation of the system into soluble and particulate components. J. Bacteriol. 66, 538-542. [7] Stanier, R.Y., Gunsalus, I.C. and Gunsalus, C.F. (1953) The enzymatic conversion of mandelic acid to benzoic acid. II. Properties of the particulate fractions. J. Bacteriol. 66, 543-547. [8] Gunsalus, C.F., Stanier, R.Y. and Gunsalus, I.C. (1953) The enzymatic conversion of mandelic acid to benzoic acid. IlL Fractionation and properties of the soluble enzymes. J. Bacteriol. 66, 548-553. [9] Caries,J. and Bourguet, M. (1957) Les acides organiques du b16. C. R. Soc. Biol. Toulouse 151, 383-387.

105 [10]

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