Isofunctional Enzymes Research Articles

Biotransformation of organic sulfides. Predictive active site models for sulfoxidation catalysed by 2,5-diketocamphane 1,2-monooxygenase and 3,6-diketocamphane 1,6-monooxygenase, enantiocomplementary enzymes from Pseudomonas putida NCIMB 10007

Growth of the bacterium Pseudomonas putida NCIMB 10007 on racemic camphor induces two enantiocomplementary diketocamphane monooxygenase isofunctional enzymes (isozymes) both able to catalyse electrophilic biooxidation of a wide range of prochiral sulfoxides to the corresponding chiral sulfoxides. Active site models to explain and predict the stereoselectivity of the sulfoxidations catalysed by both isozymes are proposed. The models are based on restrictive space descriptors derived from the optimised outcomes obtained with 23 different phenyl alkyl, benzyl alkyl, methyl alkyl and ethyl alkyl sulfides: consistency was maximised by using the same enzyme preparations throughout. The models, which are consistent with the recognised stereoselectivities of diketocamphane monooxygenase catalysed nucleophilic biooxidations of ketones to lactones, are the first to provide insight into the active site topography of FMN plus NADH-dependent Baeyer–Villiger monooxygenases. In addition, they are unique in providing a direct comparison of the active sites of two enantiocomplimentary isofunctional proteins that have evolved in the same cell line.

The molecular cloning of genes specifying some enzymes of the 3,5-xylenol degradative pathway

Enzymes for the degradation of 3,5-xylenol ofPseudomonas putida NCIB9869 are encoded on a transmissible plasmid, pRA500. Genes specifying the inducible synthesis of some enzymes encoded on pRA500, namely, citraconase (G), citramalate coenzyme-A-transferase (H), citramalyl-coenzyme-A lyase (I), maleyl-pyruvate isomerase (J) and fumarylpyruvate hydrolase (K) have been cloned on a 7.9-kbHindIII fragment into the vector pKT231 to give pRA507. Biochemical and restriction analysis of pRA507 and some of its deleted derivatives has enabled preliminary locations to be assigned to the genes encoding these enzymes. The structural genes for enzymes H and I, together with a regulatory gene controlling their expression, are located on a 1.6-kbHindIII/XhoI fragment of pRA507 and the genes for enzymes G, J and K are located within a 4.5-kbXhoI/ClaI fragment of pRA507, which lies immediately adjacent to the former fragment. Biochemical analysis of the strain carrying pRA507, of its plasmid-free derivative and of other plasmid-free derivatives of the wild-typeP. putida has provided indirect evidence for the presence of two isofunctional enzymes at three different steps of the degradative pathway. One set of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase and maleylpyruvate hydrolase is encoded on pRA500 and these enzymes are inducible only. A second set is encoded on the chromosome and these enzymes are constitutive and not further inducible. The chromosomally located genes appeared to be silent until 3,5-xylenol-negative strains were incubated for prolonged periods with compounds such asm-hydroxybenzoate orp-cresol.

Isolation and Characterization of a New Vitamin C Producing Enzyme (L-Gulono-γ-lactone Dehydrogenase) of Bacterial Origin

For the first time we found that the bacterial strain Gluconobacter oxydans DSM 4025 produced L-ascorbic acid from L-gulono-γ-lactone and therefore we isolated and purified the enzyme L-gulono-γ-lactone dehydrogenase catalyzing the oxidation reaction. G. oxydans DSM 4025 produced 8.57 and 13.9 mg of L-ascorbic acid per ml from 70.3 and 89.3 mg of L-gulono-γ-lactone per ml in the growing and resting cell systems, respectively. The enzyme was isolated from the soluble fraction of the cells of G. oxydans DSM 4025 by DEAE-cellulose, Q-Sepharose, hydroxylapatite, and Sephacryl S-300 column chromatographies. The molecular weight of the enzyme was 110, 000 and it consisted of three kinds of subunits of 61, 000, 32, 500, and 16, 5000. L-Gulono-γ-lactone was oxidized to L-ascorbic acid very rapidly in the presence of dyes, such as 2, 6-dichlorophenolindophenol or phenazine methosulfate, but oxygen was not available as an electron acceptor. The absorption spectrum of the reduced form of the native enzyme showed maxima at 416, 521, and 552 nm in the visible region, indicating the presence of cytochrome c. The enzyme isolated from G. oxydans DSM 4025 was entirely different from isofunctional enzymes of eucaryotic organisms.

Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600.

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid located dmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-known meta-cleavage pathway for catechol, and include the recently discovered meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of these meta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to the meta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations between meta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Purification and properties of a homodimeric protocatechuate 4,5-dioxygenase from Rhizobium leguminosarum

Protocatechuate 4,5-dioxygenase has been purified 100-fold from 4-hydroxybenzoate grown cells of Rhizobium leguminosarum biovar viceae. The purification yielded a homogeneous preparation with specific activity of 321 Units · mg-1 protein. The molecular weight of the homodimeric native protein was 120,000, with subunit molecular weight of 62,000. The optimum pH for catalytic activity was 9.5 and the K m for protocatechuate was 20 μM. Physical and catalytic properties of the R. leguminosarum protocatechuate 4,5-dioxygenase were different from the published characteristics of isofunctional enzymes from Pseudomonas paucimobilis and Comamonas testosteroni.

Characterization of isofunctional ring-cleaving enzymes in aniline and 3-chloroaniline degradation by Pseudomonas acidovorans CA28

During degradation of aniline and 3-chloroaniline, respectively, by Pseudomonas acidovorans CA28, selective induction of two catechol 1,2-dioxygenases (C12O) was observed. C12O I activity was the sole ring-cleaving enzyme detectable in cell-free extracts after growth on aniline, while C12O II was exclusively found after growth on 3-chloroaniline. Both enzymes were clearly differentiated by their elution behaviour on DEAE-cellulose and their substrate specificities. For C12O I high activity was demonstrable only with unsubstituted catechol, while C12O II showed preference for and high affinity towards chlorinated catechols. Therefore, evidence of different ortho-cleavage enzymes in Pseudomonas acidovorans CA28 involved in aniline and 3-chloroaniline metabolism, respectively, is indicated.

Purification and characterization of polyamine oxidase from Ascaris suum.

The interconversion of polyamines in the parasite nematode Ascaris suum by a novel type of polyamine oxidase was demonstrated. The nematode enzyme was clearly distinguishable from monoamine and diamine oxidases as well as from the mammalian polyamine oxidase, as shown by the use of the specific inhibitors pargyline, aminoguanidine and MDL 72527 respectively. All three inhibitors had no effect on the parasite polyamine oxidase, and the enzyme did not accept diamines such as putrescine, cadaverine or histamine as substrates. The parasite polyamine oxidase selectively oxidizes spermine and spermidine but not N-acetylated polyamines, whereas the mammalian tissue-type polyamine oxidase shows preference for the N-acetylated polyamines. These results suggest a regulatory function of the nematode polyamine oxidase in the degradation and interconversion of polyamines in parasite nematodes. The enzyme was purified to homogeneity by gel filtration, preparative isoelectric focusing and subsequent affinity chromatography on spermine- and berenil-Sepharose 4B. With respect to reaction type, the prosthetic group FAD, the molecular mass (66 kDa) and the contents of thiol and carbonyl groups, the polyamine oxidase from A. suum is similar to the isofunctional enzyme of mammalian tissue.

Homologies between plasmid and chromosomally-encoded benzoate-oxidizing genes in Pseudomonas putida

DNA-DNA hydridization has been used to detect homologies between the TOL plasmid-encoded gene cluster xylXYZL and the chromosomally-located benABCD genes in Pseudomonas putida; both sets of genes code for isofunctional enzymes converting benzoate and toluates into catechol and its derivatives. A DNA probe corresponding to a region downstream of xylL, however, failed to hybridize to Pseudomonas chromosomal DNA. These results support the notion that catabolic operons may evolve by successive recruitment of other genes, in this case via the juxtaposition of the benABCD gene cluster upstream of the xylE gene on TOL plasmids.

Catechol 1,2-dioxygenase isozymes in soil bacteria metabolizing aromatic compounds

Isozymes of catechol 1.2-dioxygenase (CD, EC 1.13.11.1) from 16 bacterial isolates obtained from soil and grown on aniline as a sole source of C and N have been investigated. Most of the isolates examined contained two isozymes. The 16 organisms were divided into three groups on the basis of isozyme patterns; those with two isozymes of equal enzymatic activity; those with two isozymes of differing enzymatic activity; and those with only one enzyme. Other organisms grown on benzamide as a sole source of C and N also possessed two isozymes of this enzyme. These data suggest that isofunctional enzymes of CD are extensively distributed among soil bacteria that participate in biodegrading N-containing aromatic compounds, such as aniline and benzamide.

Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600

The nucleotide sequence of a 2493 base pair (bp) region, spanning the coding regions for the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), was determined. The deduced protein sequence for HMSD is 486 amino acid residues long with an M r of 51 682. HMSD has homology with a number of aldehyde dehydrogenases from various eukaryotic sources. The deduced protein sequence for HMSH is 283 amino acids long with an M r of 30 965. The amino acid composition of this enzyme is similar to that of isofunctional enzymes from toluene and m-cresol catabolic pathways.

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.

Nucleotide sequence of lysC gene encoding the lysine-sensitive aspartokinase III of Escherichia coli K12. Evolutionary pathway leading to three isofunctional enzymes.

The lysC gene encoding the lysine-sensitive aspartokinase III of Escherichia coli K12 has been cloned and its nucleotide sequence determined. Analysis of the deduced protein sequence (449 amino acid residues) reveals that the entire sequence of aspartokinase III is homologous to the N-terminal part of the two iso- and bifunctional aspartokinase-homoserine dehydrogenases I and II of E. coli. An evolutionary pathway leading to the three molecular species present in the same organism is proposed, and the possible involvement of a highly conserved region in subunit interactions is discussed.

Degradation of phenolic compounds by the yeast Candida tropicalis HP 15. II. Some properties of the first two enzymes of the degradation pathway.

The first two enzymes of the phenol degradation pathway were determined and characterized in crude extracts from Candida tropicalis HP 15. The phenol hydroxylase (EC 1.14.13.7) was a stable NADPH2- and FAD-dependent enzyme with a pH-optimum of 7.6 to 8.0 and a broad substrate specificity. Influence of ultrasound rapidly reduced the enzyme activity. The catechol 1,2-oxygenase (EC 1.13.1.1) had a broad pH-optimum between 7.5 and 9.6 and a limited substrate specificity. Two active protein bands indicating the presence of two isofunctional enzymes were detectable after electrophoretic separation of crude and partially purified extracts on polyacrylamide gels and specific staining for catechol 1,2-oxygenase activity.

Properties of a membrane-associated benzoate-4-hydroxylase from [formula omitted], [formula omitted

Membrane fractions of benzoate-induced Rhodotorula , graminis hydroxylated benzoate in the para position as demonstrated by high-performance liquid chromatography and isotopic thin-layer chromatography. Benzoate-4-hydroxylase activity was linear as a function of enzyme concentration (washed membranes) and time, and exhibited a pH optimum of 7.6. The enzyme utilized NADPH as a source of reducing equivalents, and was stimulated by FAD. The K m's for benzoate and NADPH were calculated as ∼2.9 × 10 −5 M and ∼1.9 × 10 −5 M, respectively. The particulate nature of benzoate-4-hydroxylase together with the fact that the enzyme was pteridine-independent indicates that it is distinct from the isofunctional enzyme previously described in filamentous fungi.

Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.

Speciation, Species Delineation, and Electrophoretic Isoenzyme Patterns of the Type Strains of Kluyveromyces van der Walt emend, van der Walt

The type strains of the 20 species of the yeast genus Kluyveromyces sensu van der Walt 1970 were studied by gel electrophoresis of 11 isofunctional enzymes. These enzymes included five oxidoreductases (alcohol dehydrogenase [EC 1.1.1.1], lactate dehydrogenase [EC 1.1.1.27], malate dehydrogenase [EC 1.1.1.37], catalase [EC 1.11.1.6], and superoxide dismutase [EC 1.15.1.1]), five hydrolases (esterase [EC 3.1.1.1], alkaline phosphatase [EC 3.1.3.1], α-glucosidase [EC 3.2.1.20], β-glucosidase [EC 3.2.1.21], and exo-β-glucanase [EC 3.2.1.58]), and one lyase (aldolase [EC 4.1.2.12]). Polymorphism was evident in most of the enzymes studied. Each type strain had a unique pattern when all enzymes were considered. The results of a multivariate analysis of the electrophoretic patterns supported the division of the genus into 13 species, 2 of which comprised four and five taxa, respectively, which were recognized in 1970. Enzyme electrophoresis provided evidence that widespread gene flow does not necessarily occur between yeasts which are able to hybridize in the laboratory.

Purification and properties of a NAD-linked 1,2-propanediol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-dependent 1,2-propanediol dehydrogenase (EC 1.1.1.4) activity was detected in cell-free crude extracts of various propane-grown bacteria. The enzyme activity was much lower in 1-propanol-grown cells than in propane-grown cells of Pseudomonas fluorescens NRRL B-1244, indicating that the enzyme may be inducible by metabolites of propane subterminal oxidation. 1,2-Propanediol dehydrogenase was purified from propane-grown Ps. fluorescens NRRL B-1244. The purified enzyme fraction shows a single-protein band upon acrylamide gel electrophoresis and has a molecular weight of 760,000. It consists of 10 subunits of identical molecular weight (77,600). It oxidizes diols that possess either two adjacent hydroxy groups, or a hydroxy group with an adjacent carbonyl group. Primary and secondary alcohols are not oxidized. The pH and temperature optima for 1,2-propanediol dehydrogenase are 8.5 and 20–25 °C, respectively. The activation energy calculated is 5.76 kcal/mol. 1,2-Propanediol dehydrogenase does not catalyze the reduction of acetol or acetoin in the presence of NADH (reverse reaction). The K m values at 25 °C, pH 7.0, buffer solution for 1,2-propanediol and NAD are 2 × 10 −2 and 9 × 10 −5 m, respectively. The 1,2-propanediol dehydrogenase activity was inhibited by strong thiol reagents, but not by metal-chelating agents. The amino acid composition of the purified enzyme was determined. Antisera prepared against purified 1,2-propanediol dehydrogenase from propane-grown Ps. fluorescens NRRL B-1244 formed homologous precipitin bands with isofunctional enzymes derived from propane-grown Arthrobacter sp. NRRL B-11315, Nocardia paraffinica ATCC 21198, and Mycobacterium sp. P2y, but not from propane-grown Pseudomonas multivorans ATCC 17616 and Brevibacterium sp. ATCC 14649, or 1-propanol-grown Ps. fluorescens NRRL B-1244. Isofunctional enzymes derived from methane-grown methylotrophs also showed different immunological and catalytic properties.

Microbial oxidation of methanol: Purification and properties of formaldehyde dehydrogenase from a Pichia sp. NRRL-Y-11328

An NAD +-linked, reduced glutathione-dependent formaldehyde dehydrogenase was purified to homogeneity from soluble extracts of methanol-grown yeast, Pichia sp. Formaldehyde and methylglyoxal are oxidized in the presence of NAD + as an electron acceptor. NADP + could not replace NAD +. Other straight chain aldehydes (C 2–C 6 tested), branched-chain aldehydes (e.g., isobutyaldehyde), aromatic aldehydes (e.g., salicylal-dehyde, benzaldehyde), glutyraldehyde, glyceraldehyde, glycoaldehyde, and glyoxal-dehyde tested were not oxidized by the purified formaldehyde dehydrogenase. The product of formaldehyde oxidation by purified enzyme was demonstrated to be S-for-mylglutathione by measuring the absorption at 240 nm due to the formation of thioester of formaldehyde and reduced glutathione. The K m values for NAD +, formaldehyde, and reduced glutathione were 0.12, 0.31, and 0.16 m m, respectively, for the forward reaction at pH 8.0. The purified formaldehyde dehydrogenase also catalyzed the reduction of S-formylglutathione in the presence of NADH. Formate was not reduced by the purified enzyme. The K m values for S-formylglutathione and NADH were 0.60 and 0.25 m m, respectively, for the reverse reaction at pH 6.0. Formaldehyde dehydrogenase has a molecular weight of 84,000 as determined by gel filtration and subunit molecular weight of 41,000 as determined by sodium dodecyl sulfate-gel electrophoresis. S-Formylglutathione, a product of formaldehyde oxidation, was oxidized by the partially purified formate dehydrogenase from Pichia sp. Formate dehydrogenase has a higher affinity toward S-formylglutathione ( K m value 1.8 m m) than toward formate ( K m value 25 m m). Antiserum prepared against the purified formaldehyde dehydrogenase from Pichia sp. NRRL-Y-11328 forms strong precipitin bands with isofunctional enzymes from methanol-grown Pichia pastoris NRRL-Y-7556 and Torulopsis candida Y-11419 and weak precipitin bands with Hansenula polymorpha NRRL-Y-2214. No cross-reaction was observed with isofunctional enzyme derived from methanol-grown Kloeckera sp.

Schistosoma mansoni: Inhibition of glucosephosphate isomerase and glycolysis by sugar phosphates

Glucosephosphate isomerase (EC 5.3.1.9) of Schistosoma mansoni is inhibited competitively by a number of tetrose, pentose, and hexose phosphates with inhibitor constant ( K i) values in the range of 0.5 to 400 μ M. The most potent inhibitor is 5-phospho- d-arabinonate which resembles the cis-enediolate transition state intermediate of the reaction. These analogs were also found to be effective inhibitors of the production of lactate from glucose by suitably supplemented worm homogenates. The rank order of potency of inhibition of glycolysis was inversely related to the magnitudes of the K i values for glucosephosphate isomerase. These K i values were similar to those previously reported for mammalian glucosephosphate isomerase, suggesting similarities in the steric and electronic characteristics of the active sites of these isofunctional enzymes. This conclusion was further supported by the observed pH dependence of the inhibition by 5-phospho- d-arabinonate. Although glucosephosphate isomerase is not a rate-limiting enzyme of glycolysis, in the conventional sense, its selective inhibition could be of chemotherapeutic importance, in part because of the accumulation in glycolyzing systems of glucose 6-phosphate which is a potent feedback inhibitor of hexokinase.