Centre National de la Recherche Scientifique Unité de Recherche Associée 1461, Hôpital Necker, Paris, France.
The complete amino acid sequence of rat kidney long chain alpha-hydroxy acid oxidase has been determined by microsequencing, using a number of standard enzymatic and chemical cleavages. Peptides were purified by high pressure liquid chromatography or by gel electrophoresis followed by electrotransfer. The sequence comprises 352 residues and ends with a peroxisomal targeting sequence SRL. The present work definitely establishes that hydroxy acid oxidase is a member of the family of FMN-dependent alpha-hydroxy acid-oxidizing enzymes. The family includes lactate oxidase, short chain alpha-hydroxy acid oxidase (glycolate oxidase), flavocytochrome b2, and mandelate dehydrogenase. There are altogether 45 totally conserved positions among the six sequences known. The sequence similarities are analyzed in light of the known three-dimensional structure of flavocytochrome b2 and glycolate oxidase. It is concluded that long chain hydroxy acid oxidase should be folded as a beta 8 alpha 8 barrel and should dehydrogenate alpha-hydroxy acids according to the same chemical mechanism as other enzymes of the family, in spite of a Tyr----Phe substitution at the active site.