D175 discriminates between NADH and NADPH in the coenzyme binding site of Lactobacillus delbrueckii subsp. bulgaricus D-lactate dehydrogenase.
The NAD-dependent D-(-)-lactate dehydrogenase (D-LDH) from Lactobacillus delbrueckii subsp. bulgaricus (in short, L. bulgaricus) has been modified at position 175 by site-directed mutagenesis, changing a conserved aspartate residue into an alanine. The D175A mutant enzyme displays a 40-fold shift in coenzyme preference from NADH to NADPH. This demonstrates that D175 truly belongs to the amino acid consensus GXGXXGX(17)D (where X represents any residue) which is the signature of the coenzyme binding site of most NAD-dependent dehydrogenases.
Bernard
N
N
Unité de Génétique, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Johnsen
K
K
Holbrook
J J
JJ
Delcour
J
J
D000409:Alanine; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009243:NAD; D001224:Aspartic Acid; D009249:NADP; D007770:L-Lactate Dehydrogenase; D000409:Alanine