title: A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases.
abstract: l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K(m) and V(m) values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K(m) value was slightly improved with FBP or Mn(2+) at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T(1/2)) than the wild-type enzyme in the presence of FBP, but showed an even lower T(1/2) without FBP, where it exhibited higher activation enthalpy of inactivation (ΔH(‡)). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Arai
authors.forename: Jun
authors.initials: J
authors.lastname: Ichikawa
authors.forename: Shinta
authors.initials: S
authors.lastname: Nonaka
authors.forename: Akimasa
authors.initials: A
authors.lastname: Miyanaga
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Uchikoba
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Hayao
authors.initials: H
authors.lastname: Taguchi
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000201:enzymology; D016297:Mutagenesis, Site-Directed
chemical_list: D001426:Bacterial Proteins; D007770:L-Lactate Dehydrogenase