Cloning, Expression and Characterization of NADP-Dependent Isocitrate Dehydrogenase from Staphylococcus aureus

Abstract

The Krebs cycle dictates oxidative and reductive conditions in Staphylococcus aureus and is mainly regulated by isocitrate dehydrogenase (IDH) which plays pivotal role in the growth and pathogenesis of the bacteria. In the present study, IDH gene from S. aureus ATCC12600 was cloned in the Sma I site of pQE 30 vector; the resultant clone was named as UVIDH1. The insert in the clone was sequenced (accession number HM067707), and the sequence showed complete homology with IDH sequence of other S. aureus strains reported in the database indicating presence of single enzyme in S. aureus, and considerable sequence homology with other bacteria was observed; however, only 24% homology was found with NADP-dependent human IDH. Phylogenetically, the S. aureus IDH showed close identity with Bacillus subtilis and high degree of variability with other bacteria and human IDH. The expression of IDH in the clone UVIDH1 was induced with 1 mM IPTG, and the recombinant IDH was purified by passing through nickel metal chelate column; the purified recombinant IDH showed a single band in SDS-PAGE with a molecular weight of 40 kDa; K(m) and V(max) for isocitrate are 8.2 ± 0.28 and 525 ± 25 μM NADPH/mg/min, respectively, and for cofactor NADP 67.5 ± 2.82 μM and V(max) 50.5 ± 2.12 μM NADPH/mg/min.

Phosphorylation controls the functioning ofStaphylococcus aureusisocitrate dehydrogenase – favours biofilm formation

“…Pure IDH assay was obtained from the cytosolic fraction of UVIDH1 clone, and the enzyme assay was carried out as described earlier 13 .…”

“…The recombinant enzymes were purified by passing the cytosolic fractions of PV1 and UVP-ALP3 clones through nickel metal chelate agarose column. The pure recombinant alp was analyzed in 10% SDS-PAGE, and kinetics of purified recombinant proteins were determined as described earlier 13,31,32 .…”

“…Similarly, the phosphorylated IDH was incubated with pure alkaline phosphatase III enzyme, and this dephosphorylated IDH was used for enzyme assay 13 .…”

“…In Staphylococcus aureus, the expression of virulence factors, toxins and biofilm formation requires active functioning of TCA cycle and serine/threonine protein kinases (PknB) [5][6][7][8][9][10][11] . Krebs cycle in S. aureus is regulated by IDH whose expression regulates the carbon flow; thus, enabling the organism to adapt and form biofilms in different niches 7,9,[12][13][14] . Earlier, we have cloned, sequenced, expressed and characterized IDH gene from S. aureus ATCC12600, the sequence revealed the presence of PknB site in the active site of IDH 13 .…”

“…Krebs cycle in S. aureus is regulated by IDH whose expression regulates the carbon flow; thus, enabling the organism to adapt and form biofilms in different niches 7,9,[12][13][14] . Earlier, we have cloned, sequenced, expressed and characterized IDH gene from S. aureus ATCC12600, the sequence revealed the presence of PknB site in the active site of IDH 13 . PknB is also implicated in the cell wall and polysaccharide intercellular adhesin (PIA) synthesis, functioning of various metabolic pathways, resistance to antibacterial agents and autolysis which are all required for biofilm formation.…”