Detection of the covalent intermediate of UDP-N-acetylglucosamine enolpyruvyl transferase by solution-state and time-resolved solid-state NMR spectroscopy.
Uridine diphosphate-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase from Enterobacter cloacae catalyzes the transfer of an enolpyruvyl moiety from phosphoenolpyruvate (PEP) to the 3-hydroxyl of UDP-NAG to form enolpyruvyl UDP-NAG and inorganic phosphate. Indirect evidence for the involvement of a covalent intermediate, in which the C-2 of O-phosphothioketal moiety is attached to Cys-115, in the reaction catalyzed by UDP-NAG enolpyruvyl transferase has been reported by Wanke and Amrhein [Wanke, C., & Amrhein, N. (1993) Eur. J. Biochem. 218, 861-870]. In the enzyme from Escherichia coli, a noncovalent tetrahedral intermediate in which the C-2 of PEP is attached to the 3-OH of UDP-NAG via an ether linkage has been isolated by Marquardt et al. [Marquardt, J.L., Brown, E.D., Walsh, C.T., & Anderson, K.S. (1993) J. Am. Chem. Soc. 115, 10398-10399]. In this study, we provide direct evidence for the formation of a covalent O-phosphothioketal enzyme intermediate from UDP-NAG enolpyruvyl transferase of E. cloacae overexpressed in E. coli. The intermediate was obtained by incubation of the enzyme with [2,3-13C2]PEP and UDP-NAG and was characterized by solution-state 1D 13C and 31P NMR, 13C DEPT NMR, and 1H[13C]2D HMQC NMR spectroscopy. The 13C NMR spectra showed two coupled resonances at 29.3 and 88.7 ppm which were assigned to the C-3 and C-2 of the covalent intermediate, and the 13C DEPT confirmed that C-3 was a methyl group and C-2 was quaternary.(ABSTRACT TRUNCATED AT 250 WORDS)
Ramilo
C
C
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
Appleyard
R J
RJ
Wanke
C
C
Krekel
F
F
Amrhein
N
N
Evans
J N
JN
D019883:Alkyl and Aryl Transferases; D016972:Enterobacter cloacae / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D012996:Solutions; D014166:Transferases / Q000737:chemistry*
D012996:Solutions; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase