Abstract:
:Neisseria meningitidis α-1,4-galactosyltransferase C (LgtC) is responsible for the transfer of α-galactose from donor UDP-galactose to the lipooligosaccharide terminal acceptor lactose. Crystal structures of its substrate analogue complexes have provided key insights into the galactosyl transfer mechanism, including a hypothesized need for active site mobility. Accordingly, we have used nuclear magnetic resonance spectroscopy to probe the structural dynamics of LgtC in its apo form and with bound substrate analogues. More than the expected number of signals were observed in the methyl-TROSY spectra of apo LgtC, indicating that the protein adopts multiple conformational states. Magnetization transfer experiments showed that the predominant states, termed "a" and "b", are in equilibrium on a time scale of seconds. Their relative populations change with temperature and mutations, and only the "b" state is competent for substrate binding. For both states, relaxation dispersion studies also revealed substantial millisecond time scale motions of isoleucine side chains within and distal to the active site. Although altered, these motions were still detected in LgtC with a noncovalently bound donor analogue. A mutant, LgtC-Q189E, which forms an unexpected glycosyl-enzyme intermediate via a residue (Asp190) distal from its active site, was also investigated. Apo LgtC-Q189E did not show any enhanced motions that might account for the dramatic structural change required for the galactosylation of Asp190, yet formation of a trapped glycosyl-enzyme intermediate substantially reduced its millisecond time scale conformational mobility. Although further studies are required to link the detected motions of LgtC with its enzymatic mechanism, this work clearly demonstrates the complex structural dynamics of a model glycosyltransferase.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Chan PH,Cheung AH,Okon M,Chen HM,Withers SG,McIntosh LPdoi
10.1021/bi301317dsubject
Has Abstractpub_date
2013-01-15 00:00:00pages
320-32issue
2eissn
0006-2960issn
1520-4995journal_volume
52pub_type
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