Recognition site for the side chain of 2-ketoacid substrate in d-lactate dehydrogenase.
Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.
Ishikura
Yoshirou
Y
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510.
Tsuzuki
Shino
S
Takahashi
O
O
Tokuda
Chizuka
C
Nakanishi
Rie
R
Shinoda
Takeshi
T
Taguchi
Hayao
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D007651:Keto Acids / Q000737:chemistry*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology*; D009243:NAD; D012997:Solvents
D001426:Bacterial Proteins; D007651:Keto Acids; D012997:Solvents; D009243:NAD; D007770:L-Lactate Dehydrogenase