Crystal Structures of Pasteurella multocida Sialyltransferase Complexes with Acceptor and Donor Analogues Reveal Substrate Binding Sites and Catalytic Mechanism
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- 作者：Lisheng Ni ; Harshal A. Chokhawala ; Hongzhi Cao ; Ryan Henning ; Laura Ng ; Shengshu Huang ; Hai Yu ; Xi Chen ; Andrew J. Fisher
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 29, 2007
- 年：2007
- 卷：46
- 期：21
- 页码：6288 - 6298
- 全文大小：717K
- 年卷期：v.46,no.21(May 29, 2007)
- ISSN：1520-4995
文摘
Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologicallyimportant sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctionalPasteurella multocida sialyltransferase (
24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 Å resolutions, respectively. Ternary X-ray structures of theprotein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have beendetermined at 2.0 and 2.27 Å resolutions, respectively. This represents the first sialyltransferase structureand the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptorsimultaneously. The four structures presented here reveal that binding of the nucleotide-activated donorsugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helpsdefine the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysishave been identified. Structural and kinetic data support a direct displacement mechanism involving anoxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate theacceptor hydroxyl group.
24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 Å resolutions, respectively. Ternary X-ray structures of theprotein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have beendetermined at 2.0 and 2.27 Å resolutions, respectively. This represents the first sialyltransferase structureand the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptorsimultaneously. The four structures presented here reveal that binding of the nucleotide-activated donorsugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helpsdefine the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysishave been identified. Structural and kinetic data support a direct displacement mechanism involving anoxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate theacceptor hydroxyl group.