Cloning, Expression and Characterization of NAD Kinase from Involved in the Formation of NADP (H): A Key Molecule in the Maintaining of Redox Status and Biofilm Formation.

BACKGROUND

has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK) and this gene of ATCC 12600 was cloned, sequenced, expressed and characterized.

MATERIALS AND METHODS

The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure.

RESULTS

The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814) revealed presence of only one kind of NADK in all strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Å observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity.

CONCLUSION

The results of this study suggest increased NADPH concentration in plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.