Cloning of the sth gene from Azotobacter vinelandii and construction of chimeric soluble pyridine nucleotide transhydrogenases

Abstract

The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Azotobacter vinelandii was cloned and sequenced. This is the third sth gene identified and further defines a new subfamily within the flavoprotein disulfide oxidoreductases. The three STHs identified all lack one of the redox active cysteines that are characteristic for this large family of enzymes, and instead they contain a conserved threonine residue at this position. The recombinant A. vinelandii enzyme was purified to homogeneity and shown to form filamentous structures different from those of Pseudomonas fluorescens and Escherichia coli STH. Chimeric STHs were constructed which showed that the C-terminal region is important for polymer formation. The A. vinelandii STH containing the C-terminal region of P. fluorescens or E. coli STH showed structures resembling those of the STH contributing the C-terminal portion of the protein. ß

Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli

“…The purified enzyme was homogeneous as judged by SDS‐gel electrophoresis (Fig. 1a), and the molecular mass of each subunit, approximately 52 kDa, is consonant with the predicted molecular weight of Ec STH (51.5 kDa) and previous reports for STHs from Azotobacter vinelandii, E. coli and Pseudomonas fluorescens (French et al , 1997; Boonstra et al , 1999, 2000b). Western blot analysis reveals a single protein band using the anti‐6 × His antibody as a probe (Fig.…”

“…A search of the KEGG Enzyme Database for enzymes with STH activity, and of GenBank using NCBI blast for sequences >40% similar to E. coli sth , reveals that the enzyme is found far beyond the Gammaproteobacteria and a few mycobacteria as first reported (Boonstra et al , 2000b). Many actinobacteria and some members of the Alpha ‐, Beta ‐, Deltaproteobacteria and Spirochaetales all contain the enzyme.…”

“…Enzyme assays were performed in 1 mL volume containing 0.1 mM NADPH, 0.1 mM thio‐NAD + and 50 mM Tris‐HCl buffer (pH 7.5) at 35 °C (French et al , 1997; Boonstra et al , 1999, 2000b). The reduction of thio‐NAD + was monitored at 400 nm with a thermostated Cary 300 UV‐Vis spectrophotometer (Varian, CA) using a molar extinction coefficient of 11 300 M −1 cm −1 .…”

“…The high cost of cofactors has spurred interest in using STH as a means to regenerate them during industrial production (Boonstra et al , 2000a; van der Donk & Zhao, 2003; Wandrey, 2004). STH has been used as a biocatalyst to regenerate cofactors in the syntheses of hydromorphone and poly(3‐hydroxybutyrate), a biodegradable polymer (Boonstra et al , 2000a; Kabir & Shimizu, 2003; Sánchez et al , 2006). STH has also been used to regenerate cofactors in an organic solvent‐based reverse micelle system (Ichinose et al , 2005) as well as in a cytochrome P450BM3‐catalyzed reaction system (Mouri et al , 2009).…”