On the mechanism underlying tellurite reduction by Aeromonas caviae ST dihydrolipoamide dehydrogenase

The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K s...

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Bibliographic Details
Published in:	Biochimie Vol. 102; pp. 174 - 182
Main Authors:	Arenas, F.A., Leal, C.A., Pinto, C.A., Arenas-Salinas, M.A., Morales, W.A., Cornejo, F.A., Díaz-Vásquez, W.A., Vásquez, C.C.
Format:	Journal Article
Language:	English
Published:	France Elsevier B.V 01-07-2014
Subjects:	Aeromonas caviae Aeromonas caviae - drug effects Dihydrolipoamide dehydrogenase Dihydrolipoamide Dehydrogenase - chemistry Dihydrolipoamide Dehydrogenase - metabolism Escherichia coli Escherichia coli - genetics Gene Expression Regulation, Bacterial LpdA Mutagenesis, Site-Directed Oxidation-Reduction Tellurite Tellurite reduction Tellurium - chemistry Tellurium - toxicity Tellurite Dihydrolipoamide dehydrogenase Aeromonas caviae LpdA Tellurite reduction
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Summary:	The dihydrolipoamide dehydrogenase (LpdA) from the tellurite-resistant bacterium Aeromonas caviae ST reduces tellurite to elemental tellurium. To characterize this NADH-dependent activity, the A. caviae lpdA gene was subjected to site-directed mutagenesis and genes containing C45A, H322Y and E354K substitutions were individually transformed into Escherichia coli Δlpd. Cells expressing the modified genes exhibited decreased pyruvate dehydrogenase, dihydrolipoamide dehydrogenase and TR activity regarding that observed with the wild type A. caviae lpdA gene. In addition, cells expressing the altered lpdA genes showed increased oxidative stress levels and tellurite sensitivity than those carrying the wild type counterpart. The involvement of Cys residues in LpdA's TR activity was analyzed using specific inhibitors that interact with catalytic cysteines and/or disulfide bridges such as aurothiomalate, zinc or nickel. TR activity of purified LpdA was drastically affected by these compounds. Since LpdA belongs to the flavoprotein family, the involvement of the FAD/NAD(P)+-binding domain in TR activity was determined. FAD removal from purified LpdA results in loss of TR activity, which was restored with exogenously added FAD. Substitutions in E354, involved in FAD/NADH binding, resulted in low TR activity because of flavin loss. Finally, changing H322 (involved in NAD+/NADH binding) by tyrosine also resulted in altered TR activity. •Dihydrolipoamide dehydrogenase from A. caviae is a tellurite reductase.•C45A, H322Y and E354K changes in A. caviae lpdA increase tellurite sensitivity.•The same substitutions cause decreased PDH, DD and TR activity.•NAD(H) and FAD binding are critical for TR activity.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0300-9084 1638-6183
DOI:	10.1016/j.biochi.2014.03.008