Asp141 and the Hydrogen-Bond Chain Asp141–Asn109–Asp33 Are Respectively Essential for GT80 Sialyltransferase Activity and Structural Stability

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Xiaoyan Chen¹, Yuanming Wang², Zhenping Ma², Na Li², Weiqing Han², Qi Zhang², Yumei Cai^1*, and Jiansong Cheng^2*

¹College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Shandong 271018, China; E-mail: caiyum@163.com

²State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300071, China; E-mail: jiansongcheng@nankai.edu.cn

^* To whom correspondence should be addressed.

Received December 18, 2014; Revision received March 4, 2015

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Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. In this study, the activity of a putative sialyltransferase (Pm0160) harboring an inherent mutation D141Y in the conserved DDG motif, which has been identified in GT52 and GT80 families, was restored by reverse mutation. More interestingly, a hydrogen-bond chain was found to form between three conserved residues (Asp141, Asn109, and Asp33) of GT80 sialyltransferases based on recently determined crystal structures. Our mutagenesis experiments demonstrated that the hydrogen-bond chain connecting the general base Asp141 with Nβ4, Nβ1, and Nα1 plays an essential role in maintaining protein structural stability other than keeping the general base Asp141 in a productive orientation for sialic acid transfer.

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KEY WORDS: sialyltransferase, hydrogen-bond chain, general base, stability, activity

DOI: 10.1134/S0006297915080131

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