Identification of novel benzoylformate decarboxylases by growth selection

A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the beta-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.     

Isolation and characterization of a benzoylformate decarboxylase and a NAD/NADP-dependent benzaldehyde dehydrogenase involved in d-phenylglycine metabolism in Pseudomonas stutzeri ST-201

Following induction with d-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD⁺/NADP⁺-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.     

Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201

Following induction with D-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD+/NADP+-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.     

Comparative characterisation of thiamin diphosphate-dependent decarboxylases

Several 2-keto acid decarboxylases catalyse an acyloin condensation-like carboligase reaction beside their physiological decarboxylase activity. Although many data concerning stability and catalytic potential of these enzymes are available, a standard evaluation under similar reaction conditions is lacking. In this comprehensive survey we assemble already published data combined with new studies of three bacterial pyruvate decarboxylases, yeast pyruvate decarboxylase, benzoylformate decarboxylase from Pseudomonas putida (BFD) and the branched-chain 2-keto acid decarboxylase from Lactococcus lactis (KdcA). The obtained results proof that the optima for activity and stability are rather similar if comparable reaction conditions are used. Although the substrate ranges of the decarboxylase reaction of the various pyruvate decarboxylases are similar as well, they differ remarkably from those of BFD and KdcA. We further show that the range of acceptable donor aldehydes for the carboligase reaction of a respective enzyme can be reliably predicted from the substrate range of decarboxylase reaction.     

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.     

Factors affecting 2-hydroxypropiophenone formation by benzoylformate decarboxylase from Pseudomonas putida

Benzoylformate (100 mM) was quantitatively converted to the acyloin compound, 2-hydroxypropiophenone (61.76 mM) and benzaldehyde (38.2 mM) by an enzyme extract from Pseudomonas putida ATCC 12633 in the presence of 1.6M acetaldehyde. Biotransformations were carried out at pH 6.0 and 30 degrees C with an incubation time of 60 min. Activity of the acyloin forming enzyme, benzoylformate decarboxylase, was 1.23 units/mL in the biotransformation mixture. Acyloin formation increased dramatically with pH in the range 4-5 and had a broad activity plateau in the pH range 5-8. A broad temperature optimum for acyloin formation was also observed in the range 20-40 degrees C.     

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.     

Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl₃. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K_m for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.     

Regulation of the mandelate pathway in Pseudomonas aeruginosa

The pathway of mandelate metabolism in Pseudomonas aeruginosa is composed of the following steps: l(+)-mandelate --> benzoylformate --> benzaldehyde --> benzoate. These three steps are unique to mandelate oxidation; the benzoate formed is further metabolized via the beta-ketoadipate pathway. The first enzyme, l(+)-mandelate dehydrogenase, is induced by its substrate. The second and third enzymes, benzoylformate decarboxylase and benzaldehyde dehydrogenase, are both induced by benzoylformate. The same benzaldehyde dehydrogenase, or one very similar to it, is also induced by beta-ketoadipate, an intermediate in the subsequent metabolism of benzoate. This dehydrogenase may also be induced by adipate or a metabolite of adipate. These conclusions have been drawn from the physiological and genetic properties of wild-type P. aeruginosa strains and from the study of mutants lacking the second and third enzyme activities.     

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal. Received: 16 March 1998 / Received revision: 20 May 1998 / Accepted: 21 May 1998     

Interaction of Pseudomonas putida ATCC 12633 and Bacteriophage gh-1 in Berea Sandstone Rock

Measurements of the passage of Pseudomonas putida ATCC 12633 and a phage-resistant mutant through Berea sandstone rock were made. When bacteriophage gh-1 was adsorbed within the rock matrix, a reduction in the passage of the susceptible but not the resistant cells through the rock was observed.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

New enzymes for biotransformations

Several novel bioprocesses that have little or no counterpart in traditional methodology have recently been reported. The stereoselective and enantioselective hydrolysis of sec-alkyl sulfate esters by alkyl sulfatases proceeds with inversion of configuration and furnishes a homochiral product mixture. Haloalcohol dehalogenases were shown to accept various non-natural nucleophiles, such as azide, cyanide and nitrite for the asymmetric opening of epoxides giving rise to the corresponding azido-, cyano-, and nitro-alcohols as non-natural products. Asymmetric carbon-carbon bond formation via the acyloin- and benzoin-reaction was successfully catalyzed in water by novel lyases, such as benzoylformate decarboxylase and benzaldehyde lyase. New methods for the production of chiral nonracemic alpha-L-amino acids and amines were recently reported. Enantioselective stereoinversion of racemic alpha-aryl- and alpha-aryloxycarboxylic acids via epimerase-catalyzed inversion led to a single stereoisomeric product from the racemate.     

Oxidative decarboxylation of mandelic acid derivative by recombinant Escherichia coli: a novel method of ethyl vanillin synthesis

The benzoylformate decarboxylase gene (mdlC) from Pseudomonas putida was expressed in Escherichia coli BL21(DE3). The recombinant strain together with E. coli/pET30a-mdlB converted (S)-3-ethoxy-4-hydroxymandelic acid (S-EMA) into ethyl vanillin without ethyl vanillin degradation. 4 g ethyl vanillin/l was obtained from 10 g EMA/l within 12 h at 30 °C. This is the first report on the biotransformation of (S)-EMA to ethyl vanillin.     

Purification and properties of pyridoxal-5''-phosphate-dependent histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes.

Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.     

Feasibility of gas/solid carboligation: conversion of benzaldehyde to benzoin using thiamine diphosphate-dependent enzymes

A carboligation was investigated for the first time as an enzymatic gas phase reaction, where benzaldehyde was converted to benzoin using thiamine diphosphate (ThDP)-dependent enzymes, namely benzaldehyde lyase (BAL) and benzoylformate decarboxylase (BFD). The biocatalyst was immobilized per deposition on non-porous support. Some limitations of the gas/solid biocatalysis are discussed based on this carboligation and it is also demonstrated that the solid/gas system is an interesting tool for more volatile products.     

The mechanism of action of hexahydro-1,3,5-triethyl-s-triazine

Summary The mechanism of antimicrobial action of hexahydro-1,3,5-triethyl-s-triazine (HHTT) was studied using the HHTT-resistant isolate,Pseudomonas putida 3-T-15², its HHTT-sensitive, novobiocin-cured derivative,P. putida 3-T-15² 11:21,P. putida ATCC 12633,Pseudomonas aeruginosa PA01 andEscherichia coli J53 (RP4). HHTT was oxidized byP. putida 3-T-15², while respiration ofP. putida 3-T-15² 11:21 was inhibited by HHTT. Chemical assays showed that HHTT released formaldehyde.P. putida 3-T-15² was highly resistant to formaldehyde, whileP. putida 3-T-15² 11:21 was highly sensitive to formaldehyde. Both HHTT and formaldehyde acted similarly to inhibit proline uptake in bacterial cells and to inhibit the synthesis of the inducible enzymes, -galactosidase and glucose-6-phosphate dehydrogenase. HHTT did not have uncoupler-like activity.P. putida 3-T-15² used either HHTT or ethylamine, a component of HHTT, as a nitrogen source for growth, but neither HHTT, ethylamine or formaldehyde served as a carbon and energy source for growth. We concluded that a major mechanism of antimicrobial action of HHTT was through its degradation product, formaldehyde.     

Immunological comparison of enzymes of the β-ketoadipate pathway

-Carboxy-cis,cis-muconate lactonizing enzyme and -carboxymuconolactone decarboxylase catalyze sequential reactions in the -ketoadipate pathway, the subunit sizes of the enzymes from Pseudomonas putida, biotype A, are 40000 and 13000, respectively. The cross reaction of antisera prepared against the enzymes was tested with the isofunctional enzymes formed by representatives of other bacterial species. Despite the differences in the subunit sizes of the enzymes, the antisera revealed the same general pattern: cross reaction was observed with the corresponding enzymes formed by other strains in the fluorescent Pseudomonas RNA homology group I and generally was not observed with enzymes from other Pseudomonas species or from other bacterial genera. Exceptions were provided by representatives of Pseudomonas cepacia. Members of this species are classified outside the fluorescent Pseudomonas RNA homology group. Nevertheless, the -carboxymuconolactone decarboxylases from these organisms formed precipitin bands with antisera prepared against the corresponding enzyme from P. putida, biotype A; the lactonizing enzymes from the two species did not appear to cross react. Immunodiffusion experiments with -carboxymuconolactone decarboxylase indicated that a common set of antigenic determinants for the enzyme is conserved among strains that have been classified together by other criteria; the relative immunological distances of the decarboxylases of each taxon from the reference P. putida, biotype A, enzyme were indicated by spurring patterns on Ouchterlony plates. These results suggested that the interspecific transfer of the structural gene for the enzyme is not a common event in Pseudomonas.Non-Standard Abbreviations CMLE -carboxy-cis,cis-muconate lactonizing enzyme (EC 5.5.1.2) - CMD -carboxymuconolactone decarboxylase (EC 4.1.1.44) - MLE cis,cis-muconate lactonizing enzyme (EC 5.5.1.1) - MI muconolactone isomerase (EC 5.3.3.4) Dedicated with affection and admiration to Professor R. Y. Stanier on his 60th birthday     

Three-Component Lysine/Ornithine Decarboxylation System in Lactobacillus saerimneri 30a

Lactic acid bacteria play a pivotal role in many food fermentations and sometimes represent a health threat due to the ability of some strains to produce biogenic amines that accumulate in foods and cause trouble following ingestion. These strains carry specific enzymatic systems catalyzing the uptake of amino acid precursors (e.g., ornithine and lysine), the decarboxylation inside the cell, and the release of the resulting biogenic amines (e.g., putrescine and cadaverine). This study aimed to identify the system involved in production of cadaverine from lysine, which has not been described to date for lactic acid bacteria. Strain Lactobacillus saerimneri 30a (formerly called Lactobacillus sp. 30a) produces both putrescine and cadaverine. The sequencing of its genome showed that the previously described ornithine decarboxylase gene was not associated with the gene encoding an ornithine/putrescine exchanger as in other bacteria. A new hypothetical decarboxylation system was detected in the proximity of the ornithine decarboxylase gene. It consisted of two genes encoding a putative decarboxylase sharing sequence similarities with ornithine decarboxylases and a putative amino acid transporter resembling the ornithine/putrescine exchangers. The two decarboxylases were produced in Escherichia coli, purified, and characterized in vitro, whereas the transporter was heterologously expressed in Lactococcus lactis and functionally characterized in vivo. The overall data led to the conclusion that the two decarboxylases and the transporter form a three-component decarboxylation system, with the new decarboxylase being a specific lysine decarboxylase and the transporter catalyzing both lysine/cadaverine and ornithine/putrescine exchange. To our knowledge, this is an unprecedented observation of a bacterial three-component decarboxylation system.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.