Rat kidney L-2-hydroxyacid oxidase. Structural and mechanistic comparison with flavocytochrome b2 from bakers' yeast

Abstract

Hydroxyacid oxidase from rat kidney is an FMN-dependent enzyme that catalyzes the oxidation of L-alpha-hydroxy acids as well as, more slowly, that of L-alpha-amino acids. We report here a modified purification method for the enzyme, which is found to possess one cofactor per subunit of Mr 39,000. Determination of its N-terminal sequence suggests the protein is homologous to spinach glycolate oxidase and baker's yeast lactate dehydrogenase. In the presence of a hydroxy acid and of bromopyruvate, under anaerobic conditions, the enzyme is found to catalyze both transhydrogenation and reductive bromide ion elimination. It had previously been observed that hydroxyacid oxidase could not catalyze chloride elimination from chlorolactate in the presence of oxygen [Cromartie, T.H., & Walsh, C.T. (1975) Biochemistry 14, 3482-3490]. The behavior of this enzyme toward halogeno substrates is therefore similar to that of baker's yeast L-lactate dehydrogenase and in part different from that of Mycobacterium smegmatis lactate oxidase and porcine kidney D-amino-acid oxidase. These findings can be rationalized on the basis of a common mechanism for all these enzymes, implying formation of a carbanion as a first step, with different rate-limiting steps in the overall reaction.

The Role of a β Barrel Loop 4 Extension in Modulating the Physical and Functional Properties of Long‐Chain 2‐Hydroxy‐Acid Oxidase Isozymes

“…For rHAO p, the flat maxima are observed at 451 -453 nm and 355-357 nm. Urban et al (1988) had determined a molar absorption coefficient of 11 700 M-' cm-' at 452 nm for the enzyme purified from rat kidney. When this figure was used for calculating the protein concentration and the flavin content was separately determined by fluorimetry (see Materials and Methods), the two estimates fell within 6 % of each other for isozyme p, and within 2 % for a.…”

“…To answer some of these questions, we started studying H A 0 as an oxidase prototype, insofar as it shows a number of interesting kinetic similarities with flavocytochrome b,, a dehydrogenase-electron transferase prototype (Lindqvist et al, 1991 ;Urban et al, 1988). We recently cloned and sequenced the cDNA encoding HA0 from rat kidney (Belmouden et al, 1993).…”

Purification and some characteristics of chicken liver L‐2‐hydroxyacid oxidase A

“…Its absorbance at 280 nm was rather low and in fair agreement with Trp content, and its molecular mass (37 760 Da), as derived from Table II, was consistent with that obtained by SDS-polyacrylamide gel electrophoresis. Since a value of 169 kDa was obtained under nondenaturing conditions (not shown), it was concluded that chicken liver L-2-HAOX A is composed of 4 apparently identical subunits as all the enzymes of the A-type so far isolated from mammals [3,7] and plants [13,20].…”

“…This was shown to originate from the existence of two isozymic forms of L-2-HAOX which were further isolated and partially characterized [3]. The rat liver L-2-HAOX (isozyme A) preferentially oxidized shortchain aliphatic 2-hydroxyacids with maximal activity towards glycolate [4,5], whereas the kidney enzyme (isozyme B) catalysed the oxidation of long-chain aliphatic or aromatic 2-hydroxyacids at the highest rates [6,7].…”

“…Among mammalian hydroxyacid oxidases, rat kidney L-2-HAOX B [3,6,7], on the one hand, and L-2-HAOX A from rat [3], human [8,9] and pig [10] liver, on the other hand, have been purified and partially characterized. In vertebrates, the enzyme is believed to be involved in the metabolic production of oxalate through the successive oxidation of glycolate and glyoxylate [11], while in green plants it is one of the key enzymes Correspondence address: A. Puigserver, C.N.R.S.-C.B.M., 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 9, France in photorespiration [12].…”