2-Methylcitrate Dehydratase,a New Enzyme Functioning at the Methylcitric Acid Cycle of Propionate Metabolism

2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase), a new enzyme functioning at the methylcitric acid cycle of propionyl-CoA oxidation, was present in the cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. The enzyme was separated from the usual aconitate hydratase (EC 4.2.1.3) of the yeast with DEAE-Sephadex A-50 column chromatography. The enzyme was able to catalyze a reversible reaction between 2-methylcitrate and 2-methyl-cis-aconitate, but showed no activity on threo-d_s-2-methylisocitrate, citrate, cis- or trans-aconitate, threo-d_s-, threo-DL- or erythro-l_s-isocitrate, DL-homocitrate or other hydroxy-acids tested.

In contrast, the other enzyme fraction separated as aconitate hydratase by chromatography showed no activity on synthetic 2-methylcitrate, but was able to catalyze strongly a reversible reaction between 2-methyl-cis-aconitate and threo-d_s-2-methylisocitrate.

From these findings, the previously proposed cycle sequence was revised at the following broken arrows: propionyl-CoA+oxaloacetate → (CoASH+) 2-methylcitrate ? 2-methyl-cis-aconitate ? threo-d_s-2-methylisocitrate → pyruvate+succinate (→→oxaloacetate).

2-Methylcitrate dehydratase showed maximum activity at pH 6.5 to 7.0 and at 25 to 40°C. The enzyme was stable at temperatures up to 40°C and at pH 6.5 to 7.5, but labile in Tris-HCl buffer. The synthesis of this enzyme was constitutive in this yeast, although it was slightly repressed by glucose.     

Regulation of Enzyme Synthesis of the Glyoxylate,the Citric Acid,and the Methylcitric Acid Cycles in Candida lipolytica

Syntheses of the key enzymes of the glyoxylate cycle, in Candida lipolytica, were highly repressed by glucose. Syntheses of the key enzymes of the methylcitric acid cycle were also slightly repressed by glucose but the degrees of repression in the syntheses of these enzymes were nearly equal to those of repression in the syntheses of several enzymes of the citric acid cycle. All enzyme syntheses repressed by glucose were derepressed during incubation with succinate as well as with n-alkanes: enzyme syntheses of the methylcitric acid cycle did not necessitate the addition of propionate or odd-carbon n-alkanes. The enzymes of the methylcitric acid cycle seem to be constitutive, similarly as those of the citric acid cycle.

In the parent strain, the respective enzyme levels of the cells grown on an odd-numbered n-alkane were similar to those of the cells grown on an even-numbered n-alkane. But in the mutant strain lacking 2-methylisocitrate lyase, the cells grown on the odd-numbered alkane contained aconitate hydratase, NADP-Iinked isocitrate dehydrogenase, isocitrate lyase, 2- methylcitrate synthase and 2-methylaconitate hydratase all at higher levels than the cells grown on the even-numbered alkane. Both the parent cells and the mutant cells grown on the same carbon source contained at individually similar levels of the following six enzymes; citrate synthase, NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, and malate synthase. The pleiotropic changes of enzyme activities in the mutant cells grown on the odd-numbered alkane seem to be ascribable to direct or indirect stimulation caused by threo-d_s-2-methylisocitric acid accumulation.     

l-THREONINE DEAMINASE IN MARINE PLANKTONIC ALGAE. II. DISULFIDE AND SULFHYDRYL GROUP REQUIREMENTS OF ENZYME ACTIVITY IN TWO CRYPTOPHYTES1

The “biosynthetic”l threonine (deaminating) dehydratase of 2 cryptophytes (Chroomonas salina and Hemiselmis virescens) showed sensitive inhibition from all thiols tested (dithiothreitol, cysteine, etc.) but no effect from ascorbic acid or reduced NAD. By contrast, the enzyme activities from 5 noncryptophyceaen unicellular algae (2 cyanophytes, 1 rhodophyte, 1 diatom, 1 chlorophyte) were generally not affected by any of these reagents. The thiol reagent inhibition of the cryptophyte enzymes (1) achieved saturation with 60–70% reduction in activity, (2) was considerably reduced by pretreatment of the enzymes with l -threonine and l -isoleucine, and (3) was partially reversed by subsequent treatment with arsenite and exposure to air. It was deduced that such inhibitions were caused by thiol-specific reduction of enzyme-protein disulfide groups essential for the full expression of activity and that these groups were susceptible to ready reductive cleavage and oxidative restoration. This disulfide requirement, unique to the cryptophytes, may be the first recorded case of such a property of threonine dehydratase from all forms of life hitherto studied. The additional activity requirement of the cryptophyte enzymes for sulfhydryl groups (which requirement was common to all the algal enzymes) was confirmed (1) by the study of their sensitivity to inhibition from mercurials and disulfide-sulfhydryl exchanging reagents, and (2) by the partial reversal of these inhibitions from subsequent treatment with dithio-threitol. Both cryptophyte enzymes were densitized to feedback inhibition from l -isoleucine by prior exposure to subinhibitory concentrations of HgCl₂ or dithiodipyridine.     

3-Dehydroshikimate dehydratase in mung bean cultured cells

Summary The activity of 3-dehydroshikimate dehydratase was detected in an extract prepared from cells of mung bean (Vigna mungo) that had been cultured in the presence of shikimate while such activity was not detectable in an extract prepared from cells cultured without shikimate. The enzyme was partially purified and characterized. The maximum activity of the enzyme was observed at pH 7.4. The activity was inhibited to a small extent by EDTA and sulfhydryl inhibitors. The partially purified enzyme was sensitive to thermal denaturation but was stabilized by Mg²⁺ ions. These results suggest that 3-dehydroshikimate dehydratase might be induced in mung bean cultured cells in the presence of shikimic acid.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DHS 3-dehydroshikimic acid - PCA protocatechuic acid - QA quinic acid - SA shikimic acid - SORase shikimate - NAEP oxidoreductase     

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.     

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.     

The metabolism of gamma-aminobutyric acid (GABA) in the lobster nervous system--glutamic decarboxylase

Abstract— The glutamic acid decarboxylase has been purified from the lobster central nervous system. Potassium ion (0-075 m ) and β-mercaptoethanol (0-025 m ) were essential for enzyme activity. Enzyme had about 60 per cent of its optimal activity in the absence of added pyridoxal phosphate. Carbonyl reagents (10^?4m -hydroxylamine or amino oxyacetic acid) would abolish this residual activity. The pH optimum of the enzyme was about 8-0. Standard Michaelis-Menten kinetics were applied to the decarboxylation of glutamate and a K_m of 0.02 m was calculated. GABA inhibited the reaction (K_i= 1.25 × 10^?3m ), but the inhibition showed anomalous behaviour when graphed by the method of Lineweaver and Burk (1934). The GABA inhibition resembled competitive inhibition, but curves rather than straight lines intersecting at a common point on the velocity axis were obtained. This effect remains unexplained. Preliminary studies failed to reveal any subunit structure of the enzyme. The sedimentation coefficient (.S_20.w) was 6-55 in a sucrose density gradient in an ultracentrifuge. This was unchanged by the addition of any of the agents that influence enzyme activity. The subcellular localization of the decarboxylase was explored in crude homogenates of lobster central nervous system prepared in various ways. The major proportion (about 90 per cent) of the enzyme activity was in the soluble fraction.‘Particulate’enzyme could be prepared, but gentle suspension of this material in buffer liberated most of the activity. A contaminant in the radioactive substrates led to the production of radioactive GABA without the simultaneous evolution of CO₂. In this case, GABA production required active enzyme but was not an exclusive property of the glutamic decarboxylase activity.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Oxidation of propionate to pyruvate in Escherichia coli. Involvement of methylcitrate dehydratase and aconitase.

The pathway of the oxidation of propionate to pyruvate in Escherichia coli involves five enzymes, only two of which, methylcitrate synthase and 2-methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)-methylcitrate to (2R,3S)-2-methylisocitrate requires a novel enzyme, methylcitrate dehydratase (PrpD), and the well-known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. AcnB was purified as 2-methylaconitate hydratase from E. coli cells grown on propionate and identified by its N-terminus. The enzyme has an apparent Km of 210 micro m for (2R,3S)-2-methylisocitrate but shows no activity with (2S,3S)-methylcitrate. On the other hand, PrpD is specific for (2S,3S)-methylcitrate (Km = 440 micro m) and catalyses in addition only the hydration of cis-aconitate at a rate that is five times lower. The product of the dehydration of enzymatically synthesized (2S,3S)-methylcitrate was designated cis-2-methylaconitate because of its ability to form a cyclic anhydride at low pH. Hence, PrpD catalyses an unusual syn elimination, whereas the addition of water to cis-2-methylaconitate occurs in the usual anti manner. The different stereochemistries of the elimination and addition of water may be the reason for the requirement for the novel methylcitrate dehydratase (PrpD), the sequence of which seems not to be related to any other enzyme of known function. Northern-blot experiments showed expression of acnB under all conditions tested, whereas the RNA of enzymes of the prp operon (PrpE, a propionyl-CoA synthetase, and PrpD) was exclusively present during growth on propionate. 2D gel electrophoresis showed the production of all proteins encoded by the prp operon during growth on propionate as sole carbon and energy source, except PrpE, which seems to be replaced by acetyl-CoA synthetase. This is in good agreement with investigations on Salmonella enterica LT2, in which disruption of the prpE gene showed no visible phenotype.     

Salmonella typhimurium LT2 Catabolizes Propionate via the 2-Methylcitric Acid Cycle

We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism in Salmonella typhimurium. Results from (13)C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the alpha-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro. (1)H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.     

meso-α,ɛ-Diaminopimelate d-Dehydrogenase: Sulfhydryl Group Modification

meso-α,?-Diaminopimelate D-dehydrogenase was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and HgCl₂. Two sulfhydryl groups were titrated per molecule in the presence and absence of 6 M guanidine hydrochloride: the enzyme contained one sulfhydryl group per subunit. Modification of the sulfhydryl groups with p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-dithiopyridine, N-ethylmaleimide, and iodoacetic acid was accompanied by a loss of enzyme activity. However, modification of sulfhydryl groups of the enzyme with cyanide did not affect the activity. Thus, the introduction of bulky or charged substituents to sulfhydryl groups decreased the catalytic activity of the enzyme, but modification of the groups with the small and uncharged group, a cyano group, did not. The sulfhydryl groups did not play an essential role in catalysis.     

Dominance and recessiveness at the protein level in mutant x wildtype crosses in Sacchaomyces cerevisiae

Summary The is ₁-locus of the yeast Saccharomyces cerevisiae is the structural gene for threonine dehydratase. is ₁-mutants require isoleucine for growth and do not have active threonine dehydratase.Interallelic complementation is frequent among is ₁-mutants. This is indicative for an aggregate or multimeric structure of yeast threonine dehydratase.Complementing and non-complementing mutants were crossed to wildtype. Properties of threonine dehydratase were assayed in crude extracts of the resulting heterozygotes.Specific activities varied considerably between full wildtype activity and a level about 10% of that. The apparent Michaelis constants were increased in many heterozygotes. This effect was probably due to the aggregation of both mutant and wildtype subunits to form a hybrid threonine dehydratase with reduced substrate affinity in addition to pure wildtype enzyme. This notion is supported by the observation in one heterozygote of two enzyme fractions with increased Michaelis constants in addition to a wildtype-like fraction.The possible formation of hybrid enzymes with normal, reduced or no activity is considered to blur gene dosage relations.A given pair of alleles in a heterozygous cell can generate a new type of enzyme with properties not encountered in the corresponding two homozygous cells. This situation is not accounted for by the classical concepts of dominant-recessive or intermediate behaviour, because the difference between the heterozygotes and the homozygotes is not necessarily only quantitativ but also qualitative.We dedicate this publication to Prof. Dr. C. Auerbach on occasion of her official retirement in admiration for her pioneer work and many contribution to genetics.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Effect of EMD-IT-5914 on Chlorophyll Synthesis in Leaves of Pennisetum typhoides Seedlings

EMD-IT-5914 (5-dimethylamino-methylene-2-oxo-4-phenyl-2,5-dihydrofurane-carbonitril-(3)) inhibited chlorophyll a formation almost completely and chlorophyll b and total carotenoids up to 80% of the control, but did not appreciably affect the activity of the enzyme system succinyl-CoA synthetase/δ-aminolevulinic acid synthetase. The activity of δ-aminolevulinic acid dehydratase was not found limiting. In contrast, the herbicide strongly inhibited the activity of porphobilinogenase, and the reaction kinetics pointed towards a non-competitive type of inhibition. The results are discussed in relation to the possible role of EMD-IT-5914 in chlorophyll biosynthesis.     

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extremehalophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by l-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, l-tryptophan inhibited activity while l-tyrosine, l-methionine, l-leucine, and l-isoleucine activated the enzyme. l-Isoleucine and l-phenylalanine were effective at M levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by -2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by l-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by l-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Towards a phylogeny of phototrophic purple sulfur bacteria — the genus Ectothiorhodospira

While most strains of heterofermentative lactobacilli and strains of Leuconostoc species contained only traces of a dehydratase reacting with glycerol or propanediol-1,2, three strains of Lactobacillus brevis and one strain of L. buchneri that metabolized glycerol readily in the presence of glucose, contained propanediol-1,2 dehydratase (EC 4.2.1.28). This cobamide requiring enzyme from L. brevis B 18 was partially purified. It reacts with the substrates propanediol-1,2, glycerol and ethanediol-1,2 with the relative activities of about 3:2:1. This ratio remained unchanged throughout the purification procedure. The substrate affinities were measured: propanediol-1,2 K _m=0.6 mM, glycerol K _m=4 mM, ethanediol-1,2 K _m=5.3 mM coenzyme B₁₂ (substrate glycerol) K _m=0.007 mM. The activity of the dehydratase was promoted by potassium or ammonium ions and inhibited by sodium, lithium, magnesium or specially manganese. The apparent molecular weight of propanediol-1,2 dehydratase was determined as M_r=180,000.Dedicated to Prof. Dr. H. G. Schlegel on behalf of his 60th birthday     

The energy-yielding reactions of Peptococcus prévotii,their behaviour on starvation and the role and regulation of threonine dehydratase

The principal energy-yielding reactions of the strict anaerobe Peptococcus prévotii comprised the fermentation of l-serine and l-threonine via the enzymes threonine dehydratase, thioclastic enzyme, phosphotransacetylase and acetate kinase.Threonine dehydratase was purified 700-fold and shown to require pyridoxal 5-phosphate as co-enzyme, and a reducing agent for optimum activity. The ratio of threonine and serine dehydratase activities was unaltered during purification. The optimum pH was 8.5 to 9.5 and isoleucine did not inhibit.Lineweaver-Burk plots were linear at l-threonine concentrations above 1.35 mM and the K _m for threonine was 2.5 mM and for serine 29 mM. Below this concentration co-operativity occurred which was not nullified by individual adenine nucleotides: Hill plots were biphasic.However, the enzyme was controlled by the adenylate energy charge in a novel manner; only at very low threonine concentrations (<1 mM) was control manifest, when a high energy charge inhibited and a low energy charge stimulated activity.During starvation for 33 hrs in phosphate buffer, pH 6.8, viability fell to zero but, of the enzymes of the energy-generating sequence, only the total units and specific activity of threonine dehydratase decreased (by 35%), which was insufficient to explain the loss of ability to generate ATP.     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

Influence of Linoleate Hydroperoxide Minor Isomers on GLC Analysis of Oleate Hydroperoxides

The distribution of the methylcitric acid cycle and the modified ^-oxidation pathway for propionate catabolism was surveyed in yeasts and filamentous fungi, mainly by comparing the activities of the key enzymes. All the six tested species of filamentous fungi belonging to five genera and 21 species of yeasts belonging to eleven genera were found to catabolize propionate through the methylcitric acid cycle, with the exception of Candida rugosa and one group of strains of C. catenulata, which catabolize propionate through the ß-oxidation pathway. From the observed diversity of propionate catabolism among closely related strains or species, it was assumed that different minor pathways evolved from universal metabolic pathways, such as the citric acid cycle and the ^-oxidation pathway for fatty acids, in later stages of an evolutionary history.