ABSTRACT
L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.
Subject(s)
Norleucine/analogs & derivatives , Animals , Catalase/chemistry , Catalase/metabolism , Cattle , D-Amino-Acid Oxidase/chemistry , D-Amino-Acid Oxidase/metabolism , Glucose 1-Dehydrogenase , Glucose Dehydrogenases/chemistry , Glucose Dehydrogenases/metabolism , Glutamate Dehydrogenase/chemistry , Glutamate Dehydrogenase/metabolism , Kidney/enzymology , Liver/enzymology , Mitosporic Fungi/enzymology , NAD/metabolism , Norleucine/chemical synthesisABSTRACT
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 Km's for benzoate and NADPH were calculated as approximately 2.9 X 10(-5) M and approximately -1.9 X 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.
Subject(s)
Mitosporic Fungi/enzymology , Oxygenases/metabolism , Rhodotorula/enzymology , Benzoates , Benzoic Acid , Kinetics , Membranes/enzymology , NADP , Oxygenases/antagonists & inhibitorsABSTRACT
Microorganisms oxidize many aromatic compounds through the dihydroxylated intermediates catechol and protocatechuate and through the beta-ketoadipate pathway. The catabolic sequences used by the yeast Rhodotorula graminis for the dissimilation of aromatic compounds were elucidated after biochemical analysis of pleiotropically negative mutant strains. Growth properties of one mutant strain revealed that benzoate-4-hydroxylase was required for the utilization of phenylalanine, mandelate, and benzoate. Analysis of benzoate-4-hydroxylase- and p-hydroxybenzoate hydroxylase-deficient mutants provided genetic evidence that benzoate was hydroxylated in the para position forming p-hydroxybenzoate. Enzyme assays and growth studies with wild-type and mutant strains of R. graminis indicated that separate and highly specific hydroxylases oxidized p-hydroxybenzoate and m-hydroxybenzoate to protocatechuate. Examination of a protocatechuate 3,4-dioxygenase-deficient mutant demonstrated the role of the protocatechuate branch of the eucaryotic beta-ketoadipate pathway for the utilization of phenylalanine, mandelate, benzoate, and m-hydroxybenzoate. Salicylate, on the other hand, was shown to be metabolized through catechol. Thus, R. graminis differs from other yeasts such as Trichosporon cutaneum and Rhodotorula mucilaginosa in that it contains both branches of the beta-ketodipate pathway.