Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.
Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.
复制标题
分选酶 A 基因错义突变对变形链球菌酶活性的影响。
DOI：
10.1186/s12903-016-0204-1
复制
发表时间：
2016-04-11
期刊：
影响因子：
2.9
通讯作者：
Lin HC
中科院分区：
文献类型：
--
作者：
Zhuang PL;Yu LX;Tao Y;Zhou Y;Zhi QH;Lin HC
变形链球菌（S.变形杆菌（mutans）是龋齿的主要病原体，转肽酶分选酶A（SrtA）在致龋性中起主要作用。srtA基因中的T168 G和G470 A错义突变可能与龋齿易感性有关，正如我们以前的研究所证明的那样。本研究旨在研究srtA基因的错义突变对S.变种人将点突变的重组变形链球菌T168 G和G470 A分选酶在表达质粒pET 32a中表达。S.以变形杆菌UA 159分选酶编码基因srtA为模板进行点突变。通过定量当底物Dabcyl-QALPNTGEE-Edans被SrtA切割时产生的荧光强度的增加来评估酶活性。基于Michaelis-Menten方程的曲线拟合计算动力学常数。表达并纯化了SrtA△N40（UA 159）和突变酶SrtA△N40（D56 E）和SrtA△N40（R157 H）。动力学分析表明，SrtA△N40（D56 E）和SrtA△N40（R157 H）的亲和力与SrtA△N40（UA 159）的亲和力基本相等，由米氏常数（Km）确定。但SrtA△N40（D56 E）的催化速率常数（kcat）和催化效率（kcat/Km）均低于SrtA△N40（R157 H）和SrtA△N40（UA 159），而SrtA△N40（R157 H）的kcat和kcat/Km值略低于SrtA△N40（UA 159）。本研究的结果表明，与S. srtA基因T168 G错义突变可能与低致龋性有关。本文的在线版本（doi：10.1186/s12903-016-0204-1）包含补充材料，可供授权用户使用。
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 (Km). However, the catalytic rate constant (kcat) and catalytic efficiency (kcat/Km) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the kcat and kcat/Km 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. The online version of this article (doi:10.1186/s12903-016-0204-1) contains supplementary material, which is available to authorized users.