title: Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.
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 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
  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).
  
  
  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.
authors.affiliation: 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.
authors.forename: P L
authors.initials: PL
authors.lastname: Zhuang
authors.affiliation: 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.
authors.forename: L X
authors.initials: LX
authors.lastname: Yu
authors.affiliation: 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.
authors.forename: Y
authors.initials: Y
authors.lastname: Tao
authors.affiliation: 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.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: 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.
authors.forename: Q H
authors.initials: QH
authors.lastname: Zhi
authors.affiliation: Department of Preventive Dentistry, Guanghua School of Stomatology, Sun Yat-Sen University, 56 Ling Yuan Road West, Guangzhou, China. lin_hc@163.net.|Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-Sen University, Guangzhou, China. lin_hc@163.net.
authors.forename: H C
authors.initials: HC
authors.lastname: Lin
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D003731:Dental Caries / Q000382:microbiology; D003733:Dental Caries Susceptibility; D007700:Kinetics; D020125:Mutation, Missense; D013295:Streptococcus mutans / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Caries; Enzyme activity; Missense mutation; Streptococcus mutans; srtA