The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis.
Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905-like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh-encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6-bisphosphate were strongly dependent on pH. The activation constant increased 700-fold, whereas the K(m) for NADH increased more than 10-fold, in the pH range 5.5-7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.
Gaspar
Paula
P
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Neves
Ana R
AR
Shearman
Claire A
CA
Gasson
Michael J
MJ
Baptista
António M
AM
Turner
David L
DL
Soares
Cláudio M
CM
Santos
Helena
H
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data
D017931:DNA Primers; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D005947:Glucose