R80N MUTANT OF NUCLEOSIDE DIPHOSPHATE KINASE FROM MYCOBACTERIUM TUBERCULOSIS

Abstract

The crystal structure of the wild-type nucleoside diphosphate kinase from Mycobacterium tuberculosis at 2.6 A resolution revealed that the intersubunit salt bridge Arg80–Asp93 contributes to the thermal stability of the hexamer (Tm = 76°C). On mutating Asp93 to Asn to break the salt bridge, the thermal stability dramatically decreased by 27.6°C. Here, on mutating Arg80 to Asn, the thermal stability also significantly decreased by 8.0°C. In the X-ray structure of the R80N mutant solved at 1.9 A resolution the salt bridge was replaced by intersubunit hydrogen bonds that contribute to the thermal stability of the hexamer. A citrate anion from the crystallization buffer was bound at the bottom of the nucleotide-binding site via electrostatic and hydrogen-bonding interactions with six conserved residues involved in nucleotide binding. Structural analysis shows that the citrate is present at the location of the nucleotide phosphate groups.