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Cloning, Expression and Characterization of NADP-Dependent Isocitrate Dehydrogenase from Staphylococcus aureus.
MedLine Citation:
PMID:  23288593     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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.
Authors:
U Venkateswara Prasad; D Vasu; Y Nanda Kumar; P Santhosh Kumar; S Yeswanth; V Swarupa; B V Phaneendra; Abhijit Chaudhary; P V G K Sarma
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-4
Journal Detail:
Title:  Applied biochemistry and biotechnology     Volume:  -     ISSN:  1559-0291     ISO Abbreviation:  Appl. Biochem. Biotechnol.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-4     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8208561     Medline TA:  Appl Biochem Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, India.
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