Molecular and Biochemical Characterization of nifHDK Genes in Klebsiella pneumoniae

Abstract

Nitrogen fixation is carried by an enzyme complex called nitrogenase which consists of two main components, a dinitrogenase that is encoded by nifD and nifK and an iron containing reductase, also called Fe protein which is encoded by nifH. Nitrogen-free medium was used to detect the ability of nitrogen fixation by Klebsiella pneumonia, then DNA was extracted and overlap extension polymerase chain reaction of nifH, nifD and nifK. To obtain nucleotide sequences of these genes, sequencing of the PCR products was one. The reverse sequence of nifH and the forward sequences of nifD and nifK were converted into amino acids using online translation tool. Homology modeling was carried out using SWISS-MODEL. The modeled amino acids sequences was validated using ERRAT and PROCHECK. The modeled sequences were reliable and of quality higher than 90%. The two subunits of Fe protein were constructed and tertiary structure was predicted together with the binding sites for prosthetic group and ADP molecule in Fe protein. The following amino acids Asp11, Lys13, Asn157, Ser158, Val183, Pro184, Arg185, Asp186, Val189, Gln190 and Glu193 seem to participate in the ADP binding. The complexity of this enzyme makes it difficult to be cloned in plants.