Cloning, Expression, Purification and Characterization of UMP Kinase from Staphylococcus aureus

Abstract

Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150 kDa and exhibited monomeric form with molecular weight of 25 kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed K(m) of 2.80 ± 0.1 μM and Vmax 51.38 ± 1.39 μM of NADH/min/mg. The enzyme exhibited cooperative kinetics with ATP as substrate, as GTP decreased this cooperativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5α. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24 Å indicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials.

Phosphorylation controls the functioning ofStaphylococcus aureusisocitrate dehydrogenase – favours biofilm formation

“…Single colony from the above plate was inoculated in brain heart infusion (BHI) and Luria-Bertani (LB) broths. These cultures were grown overnight at 37 C. After overnight incubation, the grown culture was subjected to isolation of chromosomal DNA 25 .…”

Characterization of succinate dehydrogenase flavoprotein from Staphylococcus aureus ATCC 12600

“…The expressed protein gave single band in 10% SDS-PAGE with a molecular weight of 66kDa (Figure 4). 17 The eluted protein from the gel showed similar properties with that of native enzyme indicating no isoforms of SDH are present in Staphylococcus aureus. Different bacteria have variation in their TCA cycle according to their needs.…”

“…An aliquot of the supernatant was tested for the presence of protein by subjecting it to 10% SDS-PAGE. 15,17 This purified protein activity was determined by subtracting the activity of uninduced enzyme from the purified recombinant enzyme and the kinetic parameters K M , V max , K cat for recombinant succinate dehydrogenase (rSDH) flavoprotein were determined using Hanes-Woolf plot ([S] vs [S] / V).…”

Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation

“…After overnight incubation, a single black shiny colony with distinct zone was picked and inoculated overnight in brain heart infusion (BHI) and Luria bertani (LB) broths at 37 ºC with constant agitation at 120 revolutions per minute (rpm) in environmental orbital shaker. 24 S. aureus ATCC12600 culture thus grown was used to characterize IMPDH.…”

“…IMPDH is also an important enzyme required for the colonization of S. aureus and is involved in generating energy (ATP) for increased cell wall biosynthesis, small colony variants (SCV) and increased biofilm formation (Figure 1) are the characteristic features of vancomycin resistant S. aureus (VRSA) strains. 23,24 Owing to the importance of IMPDH in S. aureus, the present study is aimed in the characterization of IMPDH of American type culture collection (ATCC) of Staphylococcus aureus 12600.…”