Cloning, sequence, and properties of the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens

Abstract

The gene encoding the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.Pyridine nucleotide transhydrogenases catalyze the transfer of reducing equivalents between NAD and NADP pools. A membrane-bound, proton-pumping transhydrogenase, specific for the 4A proton of NADH and the 4B proton of NADPH, occurs in mitochondria and in some bacteria, such as Escherichia coli, and has been studied in some detail (3,8). This enzyme couples proton import with oxidation of NADH and reduction of NADP ϩ , and its physiological role is believed to be production of NADPH for reductive biosyntheses. Less wellknown is a soluble transhydrogenase (STH) reported to occur in Pseudomonas fluorescens, Pseudomonas aeruginosa, and Azotobacter vinelandii (13). This enzyme is a flavoprotein specific for the 4B protons of both NADH and NADPH. STH is not energy dependent but is strongly inhibited by NADP ϩ , suggesting that its physiological role is the conversion of NADPH generated by peripheral catabolic pathways in these bacteria to NADH, which can be oxidized for energy generation (16). STH is remarkable for its formation of large polymers. The subunit M r is approximately 54,000, whereas the minimal active form of the P. aeruginosa enzyme has an M r of approximately 1.6 million (18). This minimal form further aggregates on isolation to form filaments of lengths exceeding 500 nm (9). The enzyme from A. vinelandii displays similar behavior, although the structure of the filaments appears to be different (15). STH also shows interesting kinetic behavior, with activity strongly activated by NADPH and 2Ј-AMP and inhibited by NADP ϩ (19). The presence of Ca 2ϩ favors activation and reduces inhibition.To gain some insight into the structural basis for the aggregation and regulation of this unusual enzyme, we sought to clone the gene encoding STH from P. fluorescens NCIMB9815, a close relative of P. aeruginosa.Purification of STH from P. fluorescens. STH activity was assayed by following the reduction of thionicotinamide adenine dinucleotide (tNAD ϩ ) at 400 nm in a reaction mixture consisting of 0.1 mM NADPH and 0.1 mM tNAD ϩ (Sigma Chemical Co.) in 50 mM Tris-HCl buffer (pH 7.0). The molar extinction coefficient of tNADH at 400 nm was taken as 11,300 liters mol Ϫ1 cm Ϫ1 (2). One unit of enzyme activity was defined as that amount of activity reducing 1 mol of tNAD ϩ per min under these conditions. STH was purified from cells of P. fluorescens NCIMB9815 according to a modification of the method of Höjeberg et al.. Cells were grown to stationary phase in 1 liter of SOB medium (14). The cells were harvested by centrifugation (5,000 ϫ g for 15 min) and resuspended in 20 ml of buffer A (50 mM Tris-HCl [pH 7.0] with 2 mM dithiothreitol). The cells were then disrupted by sonication (25 bursts ...

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

“…Other reagents were of analytical or higher grade. MDH, MR, and STH were purified from recombinant E. coli as described previously (5, 7,8). The specific activities of the enzyme preparations employed were 11 U of MDH per mg, 6 U of MR per mg, and 276 U of STH per mg.…”

“…Here we address the problems of cofactor depletion in the biological production of hydromorphone by using the soluble pyridine nucleotide transhydrogenase (STH) of Pseudomonas fluorescens (7), an enzyme which can catalyze the transfer of reducing equivalents according to the following equation:…”

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

“…Genomic DNA of A. vinelandii ATCC 478 was isolated according to Ausubel et al [6]. The 1.6-kb SacII/XhoI fragment of pSTH1 [3], harbouring the sth gene of P. £uorescens, was labelled using the ECL random prime system according to the manufacturer's protocol (Amersham). DNA was blotted onto Hybond-N+ membrane, detection was achieved with the ECL detection reagents and exposure was to Hyper¢lm-ECL (Amersham).…”

“…Cloning and sequencing of the soluble transhydrogenase gene of P. £uorescens revealed that the enzyme is related to the well-known family of £avoprotein disul¢de oxidoreductases [3]. Members of this family, such as lipoamide dehydrogenase, glutathione reductase and mercuric reductase, are active as homodimers, each monomer consisting of three domains: an N-terminal £avin binding domain, a central nicotinamide cofactor binding domain and a C-terminal domain which is involved in the dimerisation.…”

Characterization of a New Member of the Flavoprotein Disulfide Reductase Family of Enzymes from Mycobacterium tuberculosis

“…Our previous studies have shown that lpdC (Rv0462) (10) and gorA (Rv2855) (11) code for LipDH and mycothione reductase, respectively. The sth (Rv2713) gene product bears high sequence identity (ϳ42%) with several well characterized STHs (12)(13)(14). The lpdA (Rv3303c) and lpdB (Rv0794c) genes were annotated as probable LipDHs based on sequence homology to LipDHs from other organisms (15).…”