Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Zhuang, P.; Yu, L.; Tao, Y.; Zhou, Y.; Zhi, Q.; Lin, H.
• Is Part Of: BMC oral health. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 9
• Publication Date: 2016-12
• Subjects: Caries -- Missense mutation -- srtA -- Streptococcus mutans -- Enzyme activity; Oral medicine -- Periodicals; 617.522005
• DOI: 10.1186/s12903-016-0204-1
• Languages: English
• ISSNs: 1472-6831

Abstract

Abstract 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 thesrtA 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 thesrtA gene on SrtA enzyme activity inS. mutans . Methods The point mutated recombinantS.mutans T168G and G470A sortases were expressed in expression plasmid pET32a.S. mutans UA159 sortase coding genesrtA 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 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 thek cat andk cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159) . Conclusions The findings of this study indicate that the T168G missense mutation of thesrtA gene results in a significant reduction in enzymatic activity compared withS. mutans UA159, suggesting that the T168G missense mutation of thesrtA gene may be related to low cariogenicity.