Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans

BMC Oral Health

Research Article

L. X. Yu¹  Y. Zhou¹  H. C. Lin¹  Q. H. Zhi¹  Y. Tao¹  P. L. Zhuang² 

[1] 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;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;Department of Stomatology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Road West, Guangzhou, China;

关键词: Caries;    Missense mutation;    srtA;    Streptococcus mutans;    Enzyme activity;   

DOI  :  10.1186/s12903-016-0204-1

 received in 2015-08-26, accepted in 2016-04-01,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundStreptococcus 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.MethodsThe 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.ResultsSrtA△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).ConclusionsThe 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.

【 授权许可】

CC BY   
© Zhuang et al. 2016