ABSTRACT
Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts from fruit bodies.
Subject(s)
Agaricus/metabolism , Urea/analogs & derivatives , Uric Acid/metabolism , Agaricus/enzymology , Agaricus/growth & development , Allantoin/metabolism , Amidine-Lyases/antagonists & inhibitors , Amidine-Lyases/metabolism , Amidohydrolases/metabolism , Imidazoles/metabolism , Urate Oxidase/antagonists & inhibitors , Urate Oxidase/metabolism , Urea/metabolism , Urease/antagonists & inhibitors , Urease/metabolismABSTRACT
Allantoate amidohydrolase from Bacillus fastidiosus was purified 170-fold to homogeneity as judged by isoelectric focusing and nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass was estimated to be 128 kDa. The enzyme appeared to be a homodimer with a subunit molecular mass of 66 kDa. The enzyme has an isoelectric point of 5.6. Allantoate amidohydrolase is a Mn(2+)-dependent enzyme exhibiting a pH optimum around 8.8. Its Km value for allantoate was estimated to be 9 mM. Similar to other microbial allantoate amidohydrolases the enzyme can be reversibly activated and inactivated. No indication for the involvement of arginine, lysine, and cysteine residues in the catalytic action of the enzyme was obtained. Diethylpyrocarbonate strongly inhibited the enzyme activity, indicating the involvement of histidine or tyrosine residues in catalytic action. However, no recovery was obtained by treatment with hydroxylamine as would be expected if such residues were modified. The enzyme could be reversibly denatured by urea, guanidine, and sodium dodecyl sulfate.
Subject(s)
Allantoin/metabolism , Bacillus/enzymology , Ureohydrolases/isolation & purification , Diethyl Pyrocarbonate/pharmacology , Enzyme Activation , Guanidine , Guanidines/pharmacology , Hydrogen-Ion Concentration , Hydroxylamine , Hydroxylamines/pharmacology , Isoelectric Point , Kinetics , Protein Conformation , Protein Denaturation , Urea/pharmacology , Ureohydrolases/chemistry , Ureohydrolases/metabolismABSTRACT
Glyoxylate, formed as a result of allantoin degradation, is converted by Hyphomicrobium species to glycerate via tartronate semialdehyde. Glyoxylate carboligase and tartronate semialdehyde reductase, the two enzymes involved, are present only in cells grown on allantoin as nitrogen source.
Subject(s)
Allantoin/metabolism , Bacteria/metabolism , Glyceric Acids/metabolism , Glyoxylates/metabolism , Tartronates/metabolismABSTRACT
Hyphomicrobium species are able to use allantoin as a nitrogen source for growth. Allantoin is broken down to glyoxylate and ammonia by the consecutive action of allantoinase, allantoicase, ureidoglycolase and urease.
Subject(s)
Allantoin/metabolism , Amidine-Lyases , Bacteria/metabolism , Amidohydrolases/metabolism , Lyases/metabolism , Urease/metabolism , Ureohydrolases/metabolismABSTRACT
Hydroxypyruvate isomerase of Bacillus fastidiosus is a novel enzyme (Braun, W. and Kaltwasser, H. (1979) Arch. Microbiol. 121, 129-134) which catalyzes the reversible conversion of tartronate semialdehyde into hydroxypyruvate. The enzyme was purified to homogeneity. The native molecule had a molecular weight of 265 000-280 000 and was composed of six subunits with a molecular weight of 45 000. The enzyme showed optimal activity at pH 6.6-7.4 and 57 degrees C. Hydroxypyruvate isomerase is stable on heating for 10 min at 67 degrees C. The enzyme appeared to be specific for tartronate semialdehyde and hydroxypyyruvate and no cofactors were involved in the reaction. The equilibrium constant K = [tartronate semialdehyde] divided by [hydroxypyruvate] was found to be 2.5 at pH 7.1, and 30 degrees C.
Subject(s)
Aldose-Ketose Isomerases , Bacillus/enzymology , Bacterial Proteins/isolation & purification , Carbohydrate Epimerases/isolation & purification , Escherichia coli Proteins , Carbohydrate Epimerases/metabolism , CatalysisABSTRACT
Cell-free extracts prepared from Pseudomonas aeruginosa cells, cultured in a medium containing allantoin as sole nsource of carbon, nitrogen and energy and harvested in the stationary phase, contain an enzymicly inactive allantoinase-inhibitor complex. Pure inhibitor was isolated by dissociation of this complex followed by gelfiltration. The inhibitor had a molecular weight of about 5500 daltons. Association between inhibitor and allantoinase was demonstrated by gelfiltration and by polyacrylamide gel-electrophoresis. The inhibitor was unstable in the absence of 1 M urea and the inactivation was accompanied by aggregate formation and appearance of urease activity. The inhibitor was also isolated from cells containing urease but no allantoinase. It was concluded that the inhibitor is a subunit of urease. Inhibitors isolated from P. aeruginosa and P. acidovorans cells were active against both allantoinase from P. aeruginosa and allantoinase from P. acidovorans.
