Purification, Molecular Cloning, and Catalytic Activity ofSchizosaccharomyces pombe Pyridoxal Reductase

Abstract

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP ؉ , was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 ؎ 1,600. The enzyme showed a single absorption peak at 280 nm (E 1% ‫؍‬ 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank TM accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K ؉ channel ␤2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.Pyridoxal reductase (PL 1 reductase) (formerly designated as PN dehydrogenase) catalyzes reduction of PL with NADPH and oxidation of PN with NADP ϩ as the reverse reaction. The enzyme was for the first time found in a budding yeast, Saccharomyces cerevisiae, i.e. bakers' (1) and brewers' (2) yeasts. Guirard and Snell (3) have purified it to homogeneity from bakers' yeast and showed that the enzyme is a monomeric protein with the molecular weight of about 33,000. They designated the enzyme as PL reductase because of the equilibrium of the enzyme reaction lying so far to formation of PN, and the substrate specificity, molecular weight, and the monomeric structure of the enzyme. The enzyme resembled chlordecone reductase (4) in optimal pH, molecular weight, specific requirement of for NADPH, and behavior toward sulfhydryl reagents, barbital, and common ketone substrates.The chlordecone reductase from human liver belongs to family 1 of the AKR superfamily.2 The AKRs form an expanding oxidoreductase superfamily classified into seven families containing a variety of monomeric oxidoreductases such as aldehyde and aldose reductases, hydroxysteroid dehydrogenases, chalcone reductases, Shaker-related voltage-gated K ϩ channel ␤2 subunits, and aflatoxin B 1 -aldehyde reductases (5). Because the nomenclature ...

myo-Inositol Catabolism in Bacillus subtilis

“…It is unlikely, however, that IolH serves as another 2KMI dehydratase because the iolE-defective mutant did not possess this enzyme activity at all and, thus, could not grow on inositol (13). Finally, IolS was reported to be homologous to pyridoxal reductase of Schizosaccharomyces pombe (28). The corresponding gene was cloned and expressed in E. coli, and this actually showed some pyridoxal reductase activity.…”

Molecular Cloning, Expression, and Properties of an Unusual Aldo-Keto Reductase Family Enzyme, Pyridoxal 4-Dehydrogenase, That Catalyzes Irreversible Oxidation of Pyridoxal

“…The PCR conditions, with TaKaRa LA Taq polymerase and GC buffer I, were essentially the same as those described previously (10). The nucleotide sequence of the amplified DNA fragment (507 bp from the 5Ј-end of pld1) was determined with an Applied Biosystems 373 DNA sequencer.…”

“…PCR was performed essentially in the same way as described previously (10). The DNA fragment (about 3 kb) containing the 3Ј-end was amplified.…”

“…The K m value for pyridoxal was measured under the same conditions with the exception that A 366 was followed instead of A 340 . Kinetic parameters were determined as described previously (10).…”

“…A calibration curve was made based on the elution pattern of catalase (M r 240,000), lactate dehydrogenase (M r 140,000), alanine aminotransferase (M r 110, 000), malate dehydrogenase (M r 72,000), and cytochrome c (M r 12,400). The subunit molecular weight was measured by SDS-PAGE (13) with the molecular weight markers as described previously (10). …”