Cloning and Sequence Analysis of the Gene Encoding NiFe-hydrogenase from Klebsiella pneumoniae

Abstract

Degenerate PCR primers were designed by multiple alignment of the protein sequences of known structural genes encoding the catalytic subunits of NiFe-hydrogenases obtained from Swiss-Prot Protein Sequence Database through CLUSTAL-W software and compared for conserved sequence motifs. A 1 kb amplified PCR product was obtained from the genomic DNA of Klebsiella pneumoniae using a set of degenerate primers, and then the inverse PCR technique was used to obtain the full hydrogenase coding region. A predicted secondary structure and a 3D structural model were constructed by homology modeling and docking. On the basis of these results, it was inferred that NiFe-hydrogenase from Klebsiella pneumoniae belongs to the membrane-bound H 2 evolving hydrogenase group (Ech hydrogenase group).