The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase

Abstract

The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.

FIG. 1

Sequence of udhA and deduced amino acid sequence of STH. Nucleotides are numbered, with 1 being the A of the initiating ATG. Restriction sites indicate the insertion sites in pBluescript SK(+). Putative promoter (−35 and −10) and terminator regions are indicated. rbs, ribosome binding site.

FIG. 2

SDS-PAGE showing purification of STH from E. coli JM109/pUDHA1. Lanes 1 and 4, M_r markers; lane 2, crude extract (20 μg); lane 3, purified STH (5 μg). Numbers on the left are in thousands.

FIG. 3

UV-visible spectrum of STH. The spectrum of purified E. coli STH (1.0 mg/ml) in 50 mM Tris-HCl buffer (pH 7.0), with 5 mM DTT and 10% glycerol, was measured against a blank consisting of the same buffer. The inset shows an enlargement of the region characteristic of an oxidized flavoprotein.

FIG. 4

Sequence alignment of E. coli STH with related enzymes. The alignment was generated by the CLUSTAL W program of the Genetics Computer Group package (8), and shading was applied by BOXSHADE; the black background shows identical residues and the grey background shows similar residues, both with a threshold of 0.5. The enzymes shown are as follows (with database accession numbers in parentheses): sth_ecoli, STH from E. coli (this study); sth_psefl, STH from P. fluorescens (GenBank U91523); udha_myctu, unknown dehydrogenase from M. tuberculosis (GenBank MTCY05A6; gene name, Rv2713); dldh_psefl, dihydrolipoamide dehydrogenase from P. fluorescens (Swissprot P14128); mera_bacsr, mercuric reductase from Bacillus sp. strain RC607 (Swissprot P16171); gshr_ecoli, glutathione reductase from E. coli (Swissprot P06715); nape_entfa, NADH peroxidase from Enterococcus faecalis (Swissport P37062). *, redox-active cysteine residues; -, Rossman fold Gly-X-Gly-X-X-Gly/Ala motifs forming the FAD and NAD(P) binding sites (14, 25). The bottom line shows the secondary structure elements aligned with dldh_psefl (from 1lpf.pdb).