Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis.
Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D003065:Coenzyme A / Q000378:metabolism; D005056:Euglena gracilis / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D011773:Pyruvates; D009243:NAD; D002245:Carbon Dioxide; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D000105:Acetyl Coenzyme A; D005486:Flavin Mononucleotide; D019289:Pyruvic Acid; D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D003065:Coenzyme A