A Novel dCMP Methylase by Engineering Thymidylate Synthase

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• 作者：Sanjay Agarwalla ; Sherry LaPorte ; Lu Liu ; Janet Finer-Moore ; Robert M. Stroud ; and Daniel V. Santi
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：December 16, 1997
• 年：1997
• 卷：36
• 期：50
• 页码：15909 - 15917
• 全文大小：509K
• 年卷期：v.36,no.50(December 16, 1997)
• ISSN：1520-4995

文摘

X-ray crystal structures of binary complexes of dUMP ordCMP with the Lactobacillus caseiTS mutant N229D, a dCMP methylase, revealed that there is a stericclash between the 4-NH₂ of dCMPand His 199, a residue which normally H-bonds to the 4-O of dUMP but isnot essential for activity. Asa result, the cytosine moiety of dCMP is displaced from the active siteand the catalytic thiol is movedfrom the C6 of the substrate about 0.5 Å further than in the wild-typeTS-dUMP complex. We reasonedthat combining the N229D mutation with mutations at residue 199 whichdid not impinge on the 4-NH₂of dCMP should correct the displacements and further favor methylationof dCMP. We therefore preparedseveral TS N229D mutants and characterized their steady state kineticparameters. TS H199A/N229Dshowed a 10¹¹ change in specificity for methylation of dCMPversus dUMP. The structures of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position andorientation of bound dCMPclosely approaches that of dUMP in wild-type TS, whereas dUMP wasdisplaced from the optimal catalyticbinding site.