Zhuang P L, Yu L X, Tao Y, Zhou Y, Zhi Q H, Lin H C
Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-Sen University, 56 Ling Yuan Road West, Guangzhou, China.
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, Guangzhou, China.
BMC Oral Health. 2016 Apr 11;16:47. doi: 10.1186/s12903-016-0204-1.
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.
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.
SrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the k cat and k cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159).
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.
变形链球菌是龋齿的主要病因，转肽酶分选酶A（SrtA）在致龋性中起主要作用。如我们之前的研究所表明，srtA基因中的T168G和G470A错义突变可能与龋齿易感性有关。本研究旨在调查srtA基因的这些错义突变对变形链球菌中SrtA酶活性的影响。
点突变的重组变形链球菌T168G和G470A分选酶在表达质粒pET32a中表达。以变形链球菌UA159分选酶编码基因srtA作为点突变的模板。通过量化底物Dabcyl-QALPNTGEE-Edans被SrtA切割时产生的荧光强度增加来评估酶活性。根据米氏方程的曲线拟合计算动力学常数。
表达并纯化了SrtA△N40(UA159)以及突变酶SrtA△N40(D56E)和SrtA△N40(R157H)。动力学分析表明，根据米氏常数（Km）确定，SrtA△N40(D56E)和SrtA△N40(R157H)的亲和力与SrtA△N40(UA159)的亲和力大致相等。然而，与SrtA△N40(R157H)和SrtA△N40(UA159)相比，SrtA△N40(D56E)的催化速率常数（kcat）和催化效率（kcat/Km）降低，而SrtA△N40(R157H)的kcat和kcat/Km值略低于SrtA△N40(UA159)。
本研究结果表明，与变形链球菌UA159相比，srtA基因的T168G错义突变导致酶活性显著降低，表明srtA基因的T168G错义突变可能与低致龋性有关。
