Abstract
Staphylococcus aureus, a natural inhabitant of nasopharyngeal tract, survives mainly as biofilms. Previously we have observed that S. aureus ATCC 12600 grown under anaerobic conditions exhibited high rate of biofilm formation and l-lactate dehydrogenase activity. Thus, the concentration of pyruvate plays a critical role in S. aureus, which is primarily catalyzed by pyruvate kinase (PK). Analyses of the PK gene sequence (JN645815) revealed presence of PknB site in PK gene indicating that phosphorylation may be influencing the functioning of PK. To establish this hypothesis the pure enzymes of S. aureus ATCC 12600 were obtained by expressing these genes in PK 1 and PV 1 (JN695616) clones and passing the cytosolic fractions through nickel metal chelate column. The molecular weights of pure recombinant PK and PknB are 63 and 73 kDa, respectively. The enzyme kinetics of pure PK showed K M of 0.69 ± 0.02 µM, while the K M of PknB for stpks (stpks = NLCNIPCSALLSSDITASVNCAK) substrate was 0.720 ± 0.08 mM and 0.380 ± 0.07 mM for autophosphorylation. The phosphorylated PK exhibited 40 % reduced activity (PK = 0.2 ± 0.015 μM NADH/min/ml to P-PK = 0.12 ± 0.01 μM NADH/min/ml). Elevated synthesis of pyruvate kinase was observed in S. aureus ATCC 12600 grown in anaerobic conditions suggesting that the formed pyruvate is more utilized in the synthesis phase, supporting increased rate of biofilm formation.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-014-0248-3) contains supplementary material, which is available to authorized users.
Highlights
Staphylococcus aureus mostly derives energy from glucose catabolism through glycolysis and Krebs cycles
The pyruvate kinase (PK) gene (1.7 kb) was PCR amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in the Sma I site of pQE30 vector (Fig. 1a) in -1 frame, and the clone was named as PK 1
In S. aureus, shift of growth conditions from aerobic to anaerobic increased the expression of glycolytic enzymes, such as GapA, Eno, Pgk, and Pyk (Fuchs et al 2007); in our previous studies we have shown elevated biofilm units and lactate dehydrogenase activity when S. aureus was grown in Brain heart infusion (BHI) broth with increased concentrations of glucose (Yeswanth et al 2013)
Summary
Staphylococcus aureus mostly derives energy from glucose catabolism through glycolysis and Krebs cycles. S. aureus generates two molecules of pyruvate for every molecule of glucose consumption that reduces two molecules of NAD? ‘‘PK’’ is one of the three regulatory enzymes in glycolysis; it exhibited homotropic positive co-operativity for PEP, but not for ADP. It controls the entire glycolytic pathway by regulating the flux from Fructose-1, 6-bisphosphate (FBP) to pyruvate (Munoz and Ponce 2003). The most common form of allosteric regulation for PK is its upregulation by FBP, which increases the affinity and reduces the co-operativity of substrate binding which depends on bound divalent cations in the active site. Bound substrate and metal ions increases affinity of FBP
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