Peter J. Large

Methyl mercaptan oxidase, a key enzyme in the metabolism of methylated sulphur compounds by Hyphomicrobium EG

SUMMARY: Methyl mercaptan (MM)-oxidase was purified tenfold to near homogeneity from Hyphomicrobium EG grown on dimethyl sulphoxide. The enzyme was a monomer with an Mr value of about 40000-50000. It catalysed the formation of stoicheiometric amounts of formaldehyde, sulphide and H2O2 from MM and O2. It had a K m of 5-10 μm for MM and was strongly inhibited by substrate concentrations above 14 μm, the K i for this inhibition being 42 μm. Ethyl mercaptan and sulphide also served as substrates for the enzyme (K m 18 and 60 μm respectively), whereas methanol was not oxidized. Upon oxidation of these compounds H2O2 was formed. Although sulphide was a substrate for the enzyme, it also acted as a non-competitive inhibitor of MM oxidation (K i 90 μm). Alcohol oxidase (EC 1.1.3.13) purified from Hansenula polymorpha was also found to oxidize MM (K m 110 μm) although at a low rate. The products formed were the same as for MM oxidation by the bacterial enzyme. In contrast to MM-oxidase however, alcohol oxidase was inactive with sulphide and was not inhibited by methyl mercaptan concentrations up to 500 μm.

Activities of the enzymes of the Ehrlich pathway and formation of branched-chain alcohols in Saccharomyces cerevisiae and Candida utilis grown in continuous culture on valine or ammonium as sole nitrogen source.

Valine aminotransferase, a key enzyme in both biosynthesis and breakdown of branched-chain amino acids, showed consistently higher activity in Candida utilis grown in continuous culture than in Saccharomyces cerevisiae, while pyruvate decarboxylase and alcohol dehydrogenase, the other two enzymes of the Ehrlich pathway of branched-chain alcohol formation, were lower in activity. By spheroplast lysis, it was shown that valine aminotransferase followed the distribution of pyruvate decarboxylase in being located in the cytosol. Replacement of ammonium as nitrogen source by valine during conditions of carbon or nitrogen limitation caused increased specific activities of these three enzymes in S. cerevisiae, but (with one exception) decreased those of C. utilis. Of the metabolites accumulating in the culture medium, little or no ethanol or branched-chain alcohols were present during carbon-limited growth of either organism, but the change to nitrogen limitation resulted in increases in concentration of 20- to 100-fold in pyruvate, acetate and non-pyruvate keto acids as well as the accumulation of branched-chain alcohols in both organisms, and of ethanol, ethyl acetate and glycerol in S. cerevisiae. When valine was the limiting nitrogen source, there was an increase in non-pyruvate keto acids and a 10- to 16-fold increase in 2-methylpropanol. Total branched-chain alcohols formed under nitrogen limitation were 2-fold higher in S. cerevisiae than in C. utilis, irrespective of nitrogen source. Accumulation of branched-chain alcohols, ethanol, acetate and glycerol was also observed during carbon-limited growth of S. cerevisiae with valine as nitrogen source at dilution rates above the critical rate for transition to respirofermentative growth. Less than 70% of the valine carbon metabolized during growth of S. cerevisiae and only 15% of that used during growth of C. utilis was recovered in identified metabolic products. Even allowing for losses by volatilization during aeration, this suggests that a significant amount of the valine is being metabolized by a route or routes other than the Ehrlich pathway, possibly via the action of branched-chain 2-keto acid dehydrogenase. The molar growth yield for the nitrogen source under either carbon or nitrogen limitation was significantly lower for growth on valine than for growth on ammonium, suggesting that breakdown of valine requires more energy. It is evident that not all the enzymes involved in branched-chain amino acid metabolism in yeasts have yet been identified, nor are their interactions properly understood.

The prosthetic group of methylamine dehydrogenase from Pseudomonas AM1: Evidence for a quinone structure

The g-value and linewidth of ESR spectra of methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating) EC 1.4.99.-) and methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) are very similar. This similarity is also reflected in electron-nuclear double resonance (ENDOR) results, the coupling constants of two protons in one enzyme equalling those in the other. The presence of a third proton in the ENDOR spectrum of methylamine dehydrogenase suggests a different structure or a different kind of interaction which can be related to the finding that the resolved ROSTHETIC GROUP IS PROTEIN-BOUND. The bound prosthetic group has a high redox-potential, supporting the conclusion from the ESR and ENDOR results that it is a quinone derivative.

4-acetamidobutyrate deacetylase in the yeast Candida boidinii grown on putrescine or spermidine as sole nitrogen source and its probable role in polyamine catabolism

SUMMARY: The yeast Candida boidinii (CBS 5777, ATCC 56897) when grown on spermidine, diaminopropane, putrescine, diaminopentane, diaminohexane, acetylputrescine or 4-acetamidobutyrate as sole nitrogen source contained a deacetylase (EC 3.5.1.-) catalysing the removal of the acetyl group from N-acetyl-β-alanine, 4-acetamidobutyrate and 5-acetamidopentanoate. The enzyme was synthesized early in the exponential growth phase when C. boidinii that had been grown in medium containing glucose and ammonium was transferred to medium in which putrescine replaced ammonium. The 4-acetamidobutyrate deacetylase was partially purified 250-fold. The stoicheiometry of the reaction was established using 4-acetamidobutyrate as substrate. The enzyme had a subunit relative molecular mass (M R) of 78 500 and a M R in the range 122000 to 143000. The pH optimum was 8·0. The K m for 4-acetamidobutyrate was 0·29 mm. The enzyme was found in a number of other yeast species and was usually associated with high levels of diamine acetyltransferase and acetylputrescine oxidase. The role of this enzyme in the catabolism of di- and polyamines (including those organisms able to use these amines as carbon source) is discussed.

Putrescine breakdown in the yeast Candida boidinii: subcellular location of some of the enzymes involved and properties of two acetamidoaldehyde dehydrogenases

SUMMARY: Two acetamidoaldehyde dehydrogenases were identified in Candida boidinii grown on putrescine as sole nitrogen source with glucose as carbon source. One of them, enzyme A, although present when cells were grown on ammonium or l-lysine, increased in activity when cells were grown on putrescine or spermidine. The other, enzyme B, was absent when the putrescine was replaced by l-lysine or ammonium, but was present if the nitrogen source was spermidine or acetylputrescine. Both dehydrogenases were active with NAD+ or NADP+ as electron acceptor. Apparent K m values for 3-acetamidopropionaldehyde and 4-acetamidobutyr-aldehyde were respectively 0·83 mM and 0·041 mM for enzyme A and 0·077 mM and 0·015 mM for enzyme B. Enzyme A was competitively inhibited by chloral hydrate with a K i of 0·6 mM, whiile enzyme B was unaffected. Both enzymes were slightly (20%) stimulated by 50 mM-KCl. Although both enzymes catalysed the oxidation of a range of aldehyde substrates, and are thus both general aldehyde dehydrogenases, it is suggested that acetamidoaldehyde dehydrogenase B is more probably specifically involved in putrescine degradation. Subcellular fractionation of spheroplast lysates showed that enzyme B was cytosolic, remaining unsedimented at 100000 g, while enzyme A co-sedimented with mitochondrial marker enzymes in a sucrose density gradient. It was also shown that acetamidoalkanoate deacetylase and acetylputrescine oxidase activities, two other key enzymes in the breakdown of putrescine and spermidine, were respectively cytosolic and peroxisomal in their location in the cell.

The Oxidative Cleavage of Alkyl-Nitrogen Bonds in Micro-organisms

Abstract1. The properties of the microbial enzymes oxidatively cleaving alkyl-nitrogen bonds are reviewed.2. The enzymes fall into three classes: oxidases, dehydrogenases and mono-oxygenases.3. The role of these enzymes in the metabolism of amines and amino acids by bacteria is discussed.

Synthesis of Certain Assimilatory and Dissimilatory Enzymes during Bacterial Adaptation to Growth on Trimethylamine

Summary: During the adaptation of Pseudomonas aminovorans from growth on succinate to growth on trimethylamine, the following enzymes were synthesized in the lag phase before exponential growth on trimethylamine began: trimethylamine and dimethylamine mono-oxygenases, trimethylamine-N-oxide aldolase (demethylase), glutathione- and NAD-dependent formaldehyde dehydrogenase, dye-linked formaldehyde dehydrogenase, hydroxypyruvate reductase and N-methylglutamate dehydrogenase. Differential plots suggested that the rate of enzyme synthesis in the lag phase exceeded the rate of synthesis during exponential growth. The evidence suggests that the enzymes discussed are essential for growth on trimethylamine, while the NADPH-dependent N-methylalanine dehydrogenase is not involved.

A two-substrate kinetic study of peroxidase cationic isoenzymes in barley malt

Abstract Ten active cationic peroxidase isoenzymes were identified by polyacrylamide gel electrophoresis of extracts from malt of Triumph barley. These were partially separated on CM-Sepharose CL-6B and their properties examined. The five active peaks had different pH optima for the oxidation of 2,2′-azinobis(3-ethylbenzthiazoline 6-sulphonate) (ABTS), ranging from pH 3.25 to 3.73, and different inactivation rates at 55°, two being significantly more stable than the rest. A two-substrate kinetic study revealed a parallel pattern of double reciprocal plots for some of the active peaks, a converging pattern for others and a range of true K m values for the isoenzyme peaks varying from 76 to 710, μM for hydrogen peroxide and 2–310, μM for ABTS. The significance of the observations is discussed in relation to the known activity of peroxidase during brewery mashing.

Regulation of the key enzymes of methylated amine metabolism in Candida boidinii

Nitrogen assimilation during growth of Candida boidinii on methylated amines as sole nitrogen source involves NADP-dependent glutamate dehydrogenase. Changes in enzyme activities during the adaptation of the yeast from growth on ammonium to growth on trimethylamine were examined. No ammonia, dimethylamine or monomethylamine could be detected in the medium during growth on trimethylamine. When two methylated amines were supplied together, they were used simultaneously, although monomethylamine was metabolized more quickly than the others. When cells were grown on a low concentration of ammonium plus higher concentrations of di- or trimethylamine, the ammonium was used first. NADP-dependent glutamate dehydrogenase was the first enzyme to be derepressed, followed by methylamine oxidase and formaldehyde dehydrogenase. Di- and trimethylamine mono-oxygenase activities only appeared when the ammonium concentration fell below 0.5 mM. At this point amine utilization could be detected and no diauxic lag was observed in the growth curve. During growth on limiting ammonium, there was an increase in the activity of methylamine oxidase (150-fold) and catalase (5-fold) in the absence of any amine, but no amine mono-oxygenase activity was detected. Addition of ammonium ions to cultures growing on dimethylamine produced an immediate repression of synthesis of methylamine oxidase, NADP-dependent glutamate dehydrogenase and the two amine mono-oxygenases. An inverse correlation was found between intracellular ammonium concentration and methylamine oxidase activity. Ammonium ions also inhibited the uptake of dimethylamine or trimethylamine by washed suspensions of dimethylamine-grown cells. It is concluded that the control of methylamine oxidase and catalase and (independently) of NADP-dependent glutamate dehydrogenase is by repression of enzyme synthesis by ammonium, while expression of amine mono-oxygenases seems to require the amine to be present in the medium. Formaldehyde and formate dehydrogenases seem also to be induced by their respective substrates.

Subcellular localization and properties of partially purified dimethylamine and trimethylamine mono-oxygenase activities in Candida utilis.

By techniques involving differential centrifugation and specific precipitation with CaCl2, it was shown that dimethylamine and trimethylamine mono-oxygenase activities co-sediment with NADPH-cytochrome c reductase activity in sphaeroplast lysates of Candida utilis grown on trimethylamine as sole nitrogen source. Since the active fraction also contained low levels of cytochromes P-450 and P-420, it was concluded that the two amine mono-oxygenases are located in the smooth endoplasmic reticulum and thus end up in the microsomal fraction on cell fractionation. Ten to twenty-fold enrichment of mono-oxygenase specific activity could be achieved by separation of activity from soluble protein by centrifugation or gel filtration. Cell-free extracts prepared in the absence of FAD showed only very low mono-oxygenase activity for either substrate. Some activity could be restored by addition of flavin nucleotides: there was a fivefold stimulation by FAD and a fourfold stimulation by FMN. All trimethylamine mono-oxygenase activity was lost when a partially purified preparation containing both activities was incubated for more than 24 h at 0 degrees C, suggesting that separate enzymes are responsible for the oxidation of secondary and tertiary amines. The enzyme preparation oxidized a wide range of secondary alkylamines up to dibutylamine and tertiary alkylamines up to tributylamine. Primary amines, choline, di- and triethanolamine, spermine, spermidine and substituted anilines were not oxidized. NADH had a lower apparent Km value and higher Vmax value than NADPH. Secondary and tertiary alkylamines containing more than one kind of alkyl group gave more than one kind of aldehyde on oxidation. Stoicheiometry determinations showed a consumption of 1 mol NAD(P)H and 1 mol O2 per mol aldehyde formed. Carbon monoxide, cyanide, proadifen hydrochloride (SKF 525-A), mercurials and mercaptoethanol all inhibited both activities.