Nuclear Magnetic Resonance Spectral Assignments of 伪-1,4-Galactosyltransferase LgtC from Neisseria meningitidis: Substrate Binding and Multiple Conformational States

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• 作者：Patrick H. W. Chan ; Sophie Weissbach ; Mark Okon ; Stephen G. Withers ; Lawrence P. McIntosh
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 16, 2012
• 年：2012
• 卷：51
• 期：41
• 页码：8278-8292
• 全文大小：908K
• 年卷期：v.51,no.41(October 16, 2012)
• ISSN：1520-4995

文摘

Lipopolysaccharide 伪-1,4-galactosyltransferase C (LgtC) from Neisseria meningitidis is responsible for a key step in lipooligosaccharide biosynthesis involving the transfer of 伪-galactose from the sugar donor UDP-galactose to a terminal acceptor lactose. Crystal structures of the complexes of LgtC with Mn²⁺ and the sugar donor analogue UDP-2-deoxy-2-fluorogalactose in the absence and presence of the sugar acceptor analogue 4鈥?deoxylactose provided key insights into the galactosyl-transfer mechanism. Combined with kinetic analyses, the enzymatic mechanism of LgtC appears to involve a 鈥渇ront-side attack鈥?S_Ni-like mechanism with a short-lived oxocarbenium鈥損hosphate ion pair intermediate. As a prerequisite for investigating the required roles of structural dynamics in this catalytic mechanism by nuclear magnetic resonance techniques, the transverse relaxation-optimized amide ¹⁵N heteronuclear single-quantum correlation and methyl ¹³C heteronuclear multiple-quantum correlation spectra of LgtC in its apo, substrate analogue, and product complexes were partially assigned. This was accomplished using a suite of complementary spectroscopic approaches, combined with selective isotopic labeling and mutagenesis of all the isoleucine residues in the protein. Only 70% of the amide signals could be detected, whereas more than the expected number of methyl signals were observed, indicating that LgtC adopts multiple interconverting conformational states. Chemical shift perturbation mapping provided insights into substrate and product binding, including the demonstration that the sugar donor analogue (UDP-2FGal) associates with LgtC only in the presence of a metal ion (Mg²⁺). These spectral assignments provide the foundation for detailed studies of the conformational dynamics of LgtC.