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High levels of active glycolate oxidase from spinach (GO) and active catalase T from Saccharomyces cerevisiae (catT) have been co-produced in the methylotrophic yeast Pichia pastoris (Pp). In sequential rounds of transformation using two selectable markers, multiple copies of the genes encoding GO and catT were integrated into the Pp chromosome under control of the methanol inducible alcohol oxidase I promoter, resulting in a strain designated MSP8.6. MSP8.6 is a second-generation biocatalyst used for the conversion of glycolate to glyoxylate in the presence of a reaction component which inhibits endogenous Pp catalase. This work demonstrates a significant advance in the utility of recombinant Pp for commercial bioprocess development.

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The methylotrophic yeast Hansenula polymorpha has been developed as an efficient production system for heterologous proteins. The system offers the possibility to cointegrate heterologous genes in anticipated fixed copy numbers into the chromosome. As a consequence co-production of different proteins in stoichiometric ratios can be envisaged. This provides options to design this yeast as an industrial biocatalyst in procedures where several enzymes are required for the efficient conversion of a given inexpensive compound into a valuable product. To this end recombinant strains have been engineered with multiple copies of expression cassettes containing the glycolate oxidase (GO) gene from spinach and the catalase T (CTT1) gene from S. cerevisiae. The newly created strains produce high levels of the peroxisomal glycolate oxidase and the cytosolic catalase T. The strains efficiently convert glycolate into glyoxylic acid, oxidizing the added substrate and decomposing the peroxide formed during this reaction into water and oxygen.

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The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity.

RESUMEN

Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxylate. We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter. Under fermentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein. This recombinant Pp strain was used as a whole-cell biocatalyst for conversion of glycolic acid to glyoxylic acid.

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A variety of methods for the immobilization of glycolate oxidase have been examined for the preparation of a catalyst for the oxidation of glycolic acid to glyoxylic acid. The co-immobilization of glycolate oxidase and catalase on oxirane acrylic beads produced a catalyst which was stable to the reaction conditions used for the oxidation, where glycolic acid and oxygen are reacted in aqueous solution in the presence of the immobilized enzyme catalyst and ethylenediamine. Under optimum reaction conditions, 99% yields of glyoxylic acid were obtained at greater than 99% conversion of glycolic acid, and the recovery and reuse of the co-immobilized enzyme catalyst was demonstrated.

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S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 6444]. After reduction of the alkylated enzyme with NaBH4, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3-hydroxypropyl)Cys]-Gly-Val-Ile-Ser-Pro - Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the alpha-subunit [Anton, D. L., & Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822]. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040] and lies in the only sequence conserved between rat liver and E. coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J. Biol. Chem. 263, 17040-17049]. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism.

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Adenosylmethionine decarboxylase catalyzes one of the first committed steps in polyamine biosynthesis. It is a member of a small class of decarboxylases that use a pyruvovyl prosthetic group rather than the more common pyridoxal cofactor. We have recently shown that AdoMet decarboxylase from E. coli is composed of stoichiometric amounts of two types of subunits; alpha (Mr = 19,000), and beta (Mr = 14,000). The NH2-terminal of the alpha subunit is blocked by the pyruvoyl group and can be sequenced only after reductive amination, which converts this to an alanine residue. The beta subunit, on the other hand, has an unblocked NH2-terminal and sequences normally. The molecular weight of the holoenzyme, estimated by gel filtration, is 136,000 suggesting that the enzyme is an alpha 4 beta 4 octamer. AdoMet decarboxylase undergoes a time dependent inactivation during turnover. The mechanism of this inactivation involves a transamination from the product, decarboxylated AdoMet, and the pyruvoyl group generating an NH2-terminal alanine. The nascent product aldehyde then eliminates methylthioadenosine, resulting in the formation of acrolein, which covalently labels the alpha subunit. How this mechanism may explain AdoMet decarboxylase turned over, and how AdoMet decarboxylase inhibitors can affect its half life will be discussed.

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S-Adenosylmethionine decarboxylase, a pyruvoyl-containing decarboxylase, is inactivated in a time-dependent process under turnover conditions. The inactivation is dependent on the presence of both substrate and Mg2+, which is also required for enzyme activity. The rate of inactivation is dependent on the concentration of substrate and appears to be saturable. Inactivation by [methionyl-3,4-14C]-adenosylmethionine results in stoichiometric labeling of the protein. In contrast, when either S-[methyl-3H]adenosylmethionine or [8-14C]adenosylmethionine is used, there is virtually no incorporation of radioactivity. Automated Edman degradation of the alpha (pyruvoyl-containing) subunit reveals that substrate inactivation results in the conversion of the pyruvoyl group to an alanyl residue. These data suggest a mechanism of inactivation which involves the transamination of the nascent product to the pyruvoyl group, followed by the elimination of methylthioadenosine and the generation of a 2-propenal equivalent which could undergo a Michael addition to the enzyme. This is the first evidence for a transamination mechanism for substrate inactivation of a pyruvoyl enzyme.

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S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.

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Aminopropyltransferases are key enzymes in the biosynthesis of the polyamines spermidine and spermine. A procedure is described for assaying these enzymes by differential charcoal adsorption of 14C-labeled decarboxylated adenosylmethionine substrate from the labeled polyamine product. This assay is linear with time and enzyme concentration, and is suitable for use with a variety of amine acceptors. This procedure has the advantage, over those previously used, that it is extremely rapid yet very sensitive.

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The gamma-glutamyl carboxylation reaction proceeds by an initial vitamin K-dependent gamma-C-H glutamyl bond cleavage and a subsequent carboxylation of the activated glutamyl residue. This system is easily uncoupled such that at low CO2 concentrations which limit the extent of carboxylation there is no effect on the rate of C-H bond cleavage. In an uncoupled system, the fate of activated glutamyl residues is to incorporate a hydrogen as demonstrated by the recovery of only unaltered glutamyl residues from digests of uncoupled reactions. In addition, in reactions carried out in tritiated, deuterated water mixtures, tritium is incorporated into the gamma positions of the glutamyl residues of peptide substrates in a vitamin K-dependent process, indicating that the hydrogen incorporated must ultimately come from solvent. These results, while not proof, put severe restraints on a radical mechanism while favoring a carbanion mechanism.

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The carbon-13 nuclear magnetic resonance spectrum of cyanocobalamin in aqueous solution has been interpreted. The assignments are based on the earlier biosynthetic studies with carbon-13-enriched precursors and on the present systematic analysis of the spectra of cyanocobalamin, cyanocobalamin lactone, cyanocobalamin lactam, cyanoepicobalamin, and several cyanocobalaminmonocarboxylic acids. The interpretation of the spectrum of cyanocobalamin greatly simplifies the structure determination of new corrinoids and should prove very helpful in future studies of these compounds. The structures of two cyanocobalamindicarboxylic acids and a cyanocobalaminmonocarboxylic acid lactone have been determined by comparing their carbon-13 magnetic resonance spectra with that of cyanocobalamin.

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Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.

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Purine-nucleoside phosphorylase (EC 2.4.2.1; purine-nucleosideorthophosphate ribosyltransferase) was purified to apparent homogeneity from Chinese hamster liver and kidneys and from V79 tissue culture cells. The enzymes from both sources appear to have identical structural and catalytic properties. A simple rapid radioisotope assay capable of detecting 0.1 nmol of product for both directions of the purine-nucleoside phosphorylase reaction is described using Bio-Rad Cu2+ Chelex in Pasteur pipet columns. At 37 degrees C the purified enzyme converts 60 mumol of guanine to guanosine per min per mg of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates that the enzyme is composed of identical subunits of 30 000 molecular weight. The native enzyme behaves as a mixture of dimers of 68 000 molecular weight and trimers of 89 000 molecular weight during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme had a sedimentation coefficient of 5.4 S, which corresponds to a molecular weight of 94 000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants of 20 muM both hypoxanthine and guanine, 35 muM form guanosine, 50 muM for inosine, and 200 muM for both ribose 1-phosphate and phosphate. During isoelectrofocusing, the enzyme forms a single major band at a pI of 5.25.

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