Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster.