2WQQ

Crystallographic analysis of monomeric CstII

Summary for 2WQQ

• Entry DOI: 10.2210/pdb2wqq/pdb
• Related: 1RO7 1RO8
• Descriptor: ALPHA-2,3-/2,8-SIALYLTRANSFERASE, CYTIDINE-5'-MONOPHOSPHATE-3-FLUORO-N-ACETYL-NEURAMINIC ACID, DI(HYDROXYETHYL)ETHER, ... (4 entities in total)
• Functional Keywords: gta, cstii, transferase, sialyltransferase, glycosyltransferase
• Biological source: CAMPYLOBACTER JEJUNI
• Total number of polymer chains: 1
• Total formula weight: 35196.02

Authors

Chan, P.H.W.,Lairson, L.L.,Lee, H.J.,Wakarchuk, W.W.,Strynadka, N.C.J.,Withers, S.G.,McIntosh, L.P. (deposition date: 2009-08-25, release date: 2009-10-27, Last modification date: 2023-12-20)

Primary citation

Chan, P.H.W.,Lairson, L.L.,Lee, H.J.,Wakarchuk, W.W.,Strynadka, N.C.J.,Withers, S.G.,Mcintosh, L.P.
NMR Spectroscopic Characterization of the Sialyltransferase Cstii from Camplyobacter Jejuni: Histidine 188 is the General Base.
Biochemistry, 48:11220-, 2009

PubMed Abstract: Cell surface glycans are often terminated by sialic acid, which is incorporated onto sugar acceptors by sialyltransferases. The crystal structure of the GT family 42 Campylobacter jejuni alpha-2,3/2,8-sialyltransferase (CstII) provides key insights into the sialyl-transfer mechanism, including tentative identification of His188 as the catalytic base. In support of this hypothesis, the CstII-H188A mutant is able to catalyze sialyl transfer from CMP-Neu5Ac to added anions such as azide and formate but not to its natural sugar acceptor lactose. Complementing this work, NMR spectroscopy was used to investigate the structure and dynamics of CstII and to measure the intrinsic pK(a) value of His188 for comparison with the pK(a) determined from the pH-dependent k(cat)/K(M) of the enzyme. By systematically introducing point mutations at the subunit interfaces, two active monomeric variants, CstII-F121D and CstII-Y125Q, were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality (1)H/(15)N-HSQC and (1)H/(13)C-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the (1)H/(15)N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a millisecond to microsecond time scale. The histidine pK(a) values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of [(13)C(epsilon1)]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK(a) value approximately 6.6 of His188 matches the apparent pK(a) value approximately 6.5 governing the pH dependence of k(cat)/K(M) for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.

PubMed: 19824695
DOI: 10.1021/BI901606N

Experimental method

X-RAY DIFFRACTION (2.25 Å)