title: Cloning and characterization of l-lactate dehydrogenase gene of Staphylococcus aureus.
abstract: Staphylococcus aureus a natural inhabitant of nasopharyngeal tract survives in the host as biofilms. In the present study S. aureus ATCC12600 grown under anaerobic conditions showed biofilm units of 0.086 as compared to 0.07 when this pathogen grown in aerobic conditions with elevated lactate formation and the same was also observed with increased biofilm units of 0.06, 0.084 and 0.167 under 0.05%, 0.1% and 0.15% glucose supplementation in BHI broth. The lactate dehydrogenase (LDH) gene which catalyzes the formation of lactate was cloned, sequenced (Accession Numbers: JN645813) and expressed in Escherichia coli DH5α. The pure recombinant LDH exhibited molecular weight of 34 kDa in SDS-PAGE and the enzyme kinetics of recombinant enzyme was found to be in the direction of lactate to pyruvate Km of 2.03 ± 0.025 μM and Kcat of 1.69 ± 0.03/min and from pyruvate to lactate Km of 1.62 ± 0.10 μM and Kcat of 1.75 ± 0.03/min. In the LDH gene sequence "LKDIMA" was found to be conserved in all Gram positive bacteria and in all human LDH isoforms even though only 39% sequence homology was observed with all human LDH isoforms. However, 92% structural homology was observed with all human LDH isoforms. The molecular docking of pyruvate and lactate to the LDH structure showed -10.298 for pyruvate while -9.297 for lactate indicating higher affinity of pyruvate compared to lactate which concurred with the elevated LDH kinetics and rate of biofilm units in anaerobic conditions.
authors.affiliation: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, Andhra Pradesh, India.
authors.forename: Sthanikam
authors.initials: S
authors.lastname: Yeswanth
authors.forename: Yellapu
authors.initials: Y
authors.lastname: Nanda Kumar
authors.forename: Uppu
authors.initials: U
authors.lastname: Venkateswara Prasad
authors.forename: Vimjam
authors.initials: V
authors.lastname: Swarupa
authors.forename: Valasani
authors.initials: V
authors.lastname: Koteswara rao
authors.forename: Potukuchi
authors.initials: P
authors.lastname: Venkata Gurunadha Krishna Sarma
mesh_terms: D020816:Amino Acid Motifs; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase
keywords: Biofilm units; K(cat); K(m); Lactate dehydrogenase; RMSD; V(max)