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Cloning, expression and characterization of NADP-dependent isocitrate dehydrogenase from Staphylococcus aureus. 2013

U Venkateswara Prasad, and D Vasu, and Y Nanda Kumar, and P Santhosh Kumar, and S Yeswanth, and V Swarupa, and B V Phaneendra, and Abhijit Chaudhary, and P V G K Sarma
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati 517507, India.

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.

UI MeSH Term Description Entries
D007521 Isocitrate Dehydrogenase An enzyme of the oxidoreductase class that catalyzes the conversion of isocitrate and NAD+ to yield 2-ketoglutarate, carbon dioxide, and NADH. It occurs in cell mitochondria. The enzyme requires Mg2+, Mn2+; it is activated by ADP, citrate, and Ca2+, and inhibited by NADH, NADPH, and ATP. The reaction is the key rate-limiting step of the citric acid (tricarboxylic) cycle. (From Dorland, 27th ed) (The NADP+ enzyme is EC 1.1.1.42.) EC 1.1.1.41. NAD Isocitrate Dehydrogenase,Isocitrate Dehydrogenase (NAD+),Isocitrate Dehydrogenase-I,Dehydrogenase, Isocitrate,Dehydrogenase, NAD Isocitrate,Isocitrate Dehydrogenase I,Isocitrate Dehydrogenase, NAD
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D013211 Staphylococcus aureus Potentially pathogenic bacteria found in nasal membranes, skin, hair follicles, and perineum of warm-blooded animals. They may cause a wide range of infections and intoxications.

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