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

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 His 188 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 His 188 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 H-1/N-15-HSQC and H-1/C-13-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the H-1/N-15-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 [C-13(epsilon l)]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK(a) value similar to 6.6 of His 188 matches the apparent pK(a) value similar to 6.5 governing the pH dependence of k(cat)/K-M for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.