d-Amino acid oxidase and catalase of detergent permeabilized Rhodotorula gracilis cells and its potential use for the synthesis of α-keto acids

The variation of kinetic parameters of D-amino acid oxidase from Rhodotorula gracilis with pH was used to gain information about the chemical mechanism of the oxidation of D-amino acids catalysed by this flavoenzyme. D-Alanine was the substrate used. The pH dependence of Vmax and Vmax/Km for alanine as substrate showed that a group with a pK value of 6.26-7.95 (pK1) must be unprotonated and a group with a pK of 10.8-9.90 (pK2) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profiles of pKi for D-aspartate as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 8.4 becomes unprotonated; this basic group was not detected in Vmax/Km profiles suggesting its involvement in binding of the β-carboxylic group of the inhibitor.

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High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

Biotechnology Letters ◽

10.1007/s10529-010-0456-9 ◽

2010 ◽

Vol 33 (3) ◽

pp. 557-563 ◽

Cited By ~ 8

Author(s):

Sandra Abad ◽

Jozef Nahalka ◽

Margit Winkler ◽

Gabriele Bergler ◽

Robert Speight ◽

...

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Acid Oxidase ◽

High Level Expression ◽

Amino Acid Oxidase ◽

Rhodotorula Gracilis ◽

High Level

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ON THE RECOMBINATION PROCESS OF APO-D-AMINO ACID OXIDASE FROM Rhodotorula gracilis

Flavins and Flavoproteins 1990 ◽

10.1515/9783110855425-033 ◽

1991 ◽

pp. 171-174

Author(s):

Paola Casalin ◽

Loredano Pollegioni ◽

Bruno Curti ◽

Mirella Pilone Simonetta

Keyword(s):

Amino Acid ◽

Recombination Process ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Rhodotorula Gracilis

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Role of renal d-amino-acid oxidase in pharmacokinetics of d-leucine

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00397.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. E160-E165 ◽

Cited By ~ 22

Author(s):

Hiroshi Hasegawa ◽

Takehisa Matsukawa ◽

Yoshihiko Shinohara ◽

Ryuichi Konno ◽

Takao Hashimoto

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Renal Mass ◽

Acid Oxidase ◽

Mass Reduction ◽

Chiral Inversion ◽

Amino Acid Oxidase ◽

Isotope Tracer ◽

Keto Acids

d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding α-keto acids, but its contribution in vivo is poorly understood because the α-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their α-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[2H7]leucine to mutant mice lacking DAO activity (ddY/DAO−) and normal mice (ddY/DAO+), elimination of d-[2H7]leucine and formation of α-[2H7]ketoisocaproic acid ([2H7]KIC) and l-[2H7]leucine in plasma were determined. The ddY/DAO− mice, in contrast to ddY/DAO+ mice, failed to convert d-[2H7]leucine to [2H7]KIC and l-[2H7]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[2H7]leucine and formation of [2H7]KIC and l-[2H7]leucine. Renal mass reduction slowed down the elimination of d-[2H7]leucine. The fraction of conversion of d-[2H7]leucine to [2H7]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[2H7]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC.

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