Interactions of reduced and oxidized nicotinamide mononucleotide with wild-type and alphaD195E mutant proton-pumping nicotinamide nucleotide transhydrogenases from Escherichia coli.
The interaction of reduced nicotinamide mononucleotide (NMNH), constituting one half of NADH, with the wild-type and alphaD195E proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated. Reduction of thio-NADP+ by NMNH was catalysed at approximately 30% of the rate with NADH. Other activities including proton pumping and the cyclic reduction of 3'-acetyl-pyridine-NAD+ by NMNH in the presence of NADP+ were more strongly inhibited. The alphaD195 residue is assumed to interact with the 2'-OH moiety of the adenosine-5'-phosphate, i.e., the second nucleotide of NADH. Mutation of this residue to alphaD195E resulted in a 90% decrease in activity with NMNH as well as NADH as substrate, suggesting that it produced global structural changes of the NAD(H) binding site. The results suggest that the NMN moiety of NADH is a substrate of transhydrogenase, and that the adenine nucleotide is not required for catalysis or proton pumping.
Hu
X
X
Department of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, Box 462, SE-405 30 Göteborg, Sweden.
Zhang
J
J
Rydström
J
J
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism*; D009537:Nicotinamide Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D017354:Point Mutation; D017494:Proton Pumps / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017494:Proton Pumps; D009537:Nicotinamide Mononucleotide; D009249:NADP; D009250:NADP Transhydrogenases