title: Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined.
abstract: The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD+-dependent L-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator D-fructose-1,6-bisphosphate (Fru-1,6-P2). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with Vmax for the reduction of pyruvate being approximately tenfold that of the value for L-lactate oxidation at pH 7.5. No deviation from Michaelis-Menten kinetics was observed in the presence of Fru-1,6-P2, while a sigmoidal response (indicative of positive cooperativity) was seen towards L-lactate without Fru-1,6-P2. Strikingly, when introduced into an lldD- strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on L-lactate as the sole carbon source.
authors.affiliation: UCD School of Biomolecular and Biomedical Sciences, Conway Institute, University College Dublin, Belfield, Dublin 4, Republic of Ireland.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Sharkey
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Maher
authors.forename: Armel
authors.initials: A
authors.lastname: Guyonvarch
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005635:Fructosediphosphates / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D009154:Mutation; D009243:NAD / Q000378:metabolism; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005635:Fructosediphosphates; D011994:Recombinant Proteins; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; C029063:fructose-1,6-diphosphate