Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae.

IF 2.2 4区 生物学
S. Bose, D. Purkait, D. Joseph, V. Nayak, R. Subramanian
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引用次数: 0

Abstract

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.
霍乱弧菌cmp - n -乙酰神经胺酸合成酶的结构和功能表征。
几种致病细菌利用唾液酸,包括宿主衍生的n -乙酰神经氨酸(Neu5Ac),至少有两种方式:它们将唾液酸用作营养来源,并通过在自身涂上Neu5Ac作为逃避宿主的策略。鉴于唾液酸在多种致病菌(包括脑膜炎奈瑟菌、流感嗜血杆菌、多杀性巴氏杆菌和霍乱弧菌)的发病和宿主肠道定植中的重要作用,唾液酸分解代谢、生物合成和掺入途径中的几种酶被认为是潜在的药物靶点。本文报道了霍乱弧菌中cmp - n -乙酰神经胺酸合成酶(CMAS)的结构和功能特征。CMAS利用CTP和唾液酸催化合成cmp -唾液酸。测定了该酶的载脂蛋白和CDP结合形式的晶体结构,从而鉴定了与CDP和不变的Asp215残基相互作用的活性位点的金属辅因子Mg2+。虽然已经对真核生物和其他细菌物种的酶的开放和封闭结构形式进行了表征,但在CDP结合后观察到霍乱弧菌CMAS (VcCMAS)的部分封闭结构,代表中间状态,本文报道。动力学数据表明,VcCMAS能够激活两种最常见的唾液酸衍生物Neu5Ac和Neu5Gc。通过对VcCMAS活性位点与真核生物和其他细菌活性位点的氨基酸序列和结构比较,揭示了与唾液酸的C5取代基相互作用的不同疏水口袋。从核苷酸(CTP)和产物(cmp -唾液酸)与VcCMAS结合的热力学特征分析提供了结合过程的基本信息。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
自引率
13.60%
发文量
0
审稿时长
3 months
期刊介绍: Acta Crystallographica Section D welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules or the methods used to determine them. Reports on new structures of biological importance may address the smallest macromolecules to the largest complex molecular machines. These structures may have been determined using any structural biology technique including crystallography, NMR, cryoEM and/or other techniques. The key criterion is that such articles must present significant new insights into biological, chemical or medical sciences. The inclusion of complementary data that support the conclusions drawn from the structural studies (such as binding studies, mass spectrometry, enzyme assays, or analysis of mutants or other modified forms of biological macromolecule) is encouraged. Methods articles may include new approaches to any aspect of biological structure determination or structure analysis but will only be accepted where they focus on new methods that are demonstrated to be of general applicability and importance to structural biology. Articles describing particularly difficult problems in structural biology are also welcomed, if the analysis would provide useful insights to others facing similar problems.
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