UBC Theses and Dissertations
Pyridine nucleotide transhydrogenase of Escherichia coli: nucleotide sequence of the pnt gene and characterization of the enzyme complex Clarke, David Morgan
Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, three clones were isolated bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into pUC13. Examination of several deletion derivatives of the resulting plasmids and subsequent treatment with exonuclease BAL31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids and by in vitro transcription/translation of pDC21. Two polypeptides of molecular weights 52,000 and 48,000 were coded by the 3.05-kilobase fragment of pDC21. Both polypeptides were required for expression of transhydrogenase activity. The transhydrogenase was purified from cytoplasmic membranes of E. coli by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, α and β, of molecular weights 52,000 and 48,000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of fluorescence of 9-aminoacridine. It was concluded that E. coli transhydrogenase acts as a proton pump which is regulated primarily by a pH gradient rather than a membrane potential. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatorily linked. [¹⁴C]Dicyclohexylcarbodiimide preferentially labelled the a subunit. The transhydrogenase-catalyzed reduction of 3-acetylpyridine adenine dinucleotide by NADPH was stimulated over three-fold by NADH. It was concluded that NADH binds to an allosteric binding site on the enzyme. The nucleotide sequences of the pntA and pntB genes, coding for the transhydrogenase αand β subunits respectively, were established. The molecular masses of 53,906 (α) and 48,667 (β) and the N-terminal sequences of the predicted polypeptides agree well with the data obtained by analysis of the purified subunits. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed for each subunit.
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