NMR Spectroscopic Characterization of the Sialyltransferase CstII from Campylobacter jejuni: Histidine 188 Is the General Base

详细信息    查看全文

• 作者：Patrick H. W. Chan ; Luke L. Lairson ; Ho Jun Lee ; Warren W. Wakarchuk ; Natalie C. J. Strynadka ; Stephen G. Withers ; Lawrence P. McIntosh
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：December 1, 2009
• 年：2009
• 卷：48
• 期：47
• 页码：11220-11230
• 全文大小：977K
• 年卷期：v.48,no.47(December 1, 2009)
• ISSN：1520-4995

文摘

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 α-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 ¹H/¹⁵N-HSQC and ¹H/¹³C-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the ¹H/¹⁵N-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 [¹³C^ε1]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK_a value 6.6 of His188 matches the apparent pK_a value 6.5 governing the pH dependence of k_cat/K_M for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.