RESUMEN
BACKGROUND: Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans. METHODS: The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. RESULTS: SrtAâ³N40(UA159) and the mutant enzymes, SrtAâ³N40(D56E) and SrtAâ³N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtAâ³N40(D56E) and SrtAâ³N40(R157H) remained approximately equal to the affinity of SrtAâ³N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtAâ³N40(D56E) were reduced compared with those of SrtAâ³N40(R157H) and SrtAâ³N40(UA159), whereas the k cat and k cat /K m values of SrtAâ³N40(R157H) were slightly lower than those of SrtAâ³N40(UA159). CONCLUSIONS: The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.