Expression,purification and biochemical characterization of N-acetylglutamate kinase gene(argB) from Streptococcus mutans,the leading pathogen of human dental caries,were studied.The argB gene was amplified by polymerase chain reaction(PCR) with S.mutans genomic DNA as template.The PCR fragments were digested and ligated into the cloning/expression vector pET28a to construct the recombinant pET28a-argB.After verified by DNA sequencing,it was transformed into expressing cells E.coli BL21(DE3).The SDS-PAGE assay on the expressing cells E.coli BL21(DE3)/pET28a-argB displayed that the argB gene could be expressed in soluable form after induction with IPTG.The target NAGK was obtained by Ni chelating chromatography and gel size-exclusion chromatography.Enzyme activity assays conformed that the key enzyme S.mutans NAGK isn't inhibited by arginine,the final product in the acetyl cyclic route of arginine biosynthesis;thus implied that there may be other regulation mechanism,so it was necessary to make further investigation into arginine biosynthesis in S.mutans.The observation of analytical gel chromatography demonstrated that the enzymatic S.mutans NAGK is monomeric,apparently which was inconsistent with the reports about that in the amino acid kinase family.

The N-acetylglutamate kinase from Streptococcus mutans was expressed in Escherichia coli in soluble form and purified to homogeneity. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method and diffracted to 2.06 A. The crystal belonged to space group P2(1)2(1)2, with unit cell parameters a = 57.19 A, b =94.76 A, c =47.58 A. The gel filtration and initial phasing results showed that the enzyme exists as a monomer, which is different from previously reported N-acetylglutamate kinases.