Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis

S. Shukla, A. Verma, I. Kajala, Antti Nyyssolä, Rwivoo Baruah, K. Katina, R. Juvonen, M. Tenkanen, A. Goyal
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引用次数: 10

Abstract

ABSTRACT The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. WcCab3-rDSR gave Km of 6.2 mM and Vm of 6.3 µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)8 triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.
乳酸菌重组葡聚糖酶Cab3的结构建模及功能分析
摘要:本研究扩增、克隆并在乳球菌中表达了糊精酵母(Weissella confusa Cab3)葡聚糖蔗糖酶基因,该基因具有4.2 kb的开放阅读框,编码1402个氨基酸。重组葡聚糖酶WcCab3-rDSR以胞外酶形式在M17培养基中表达,比活性为1.5 U/mg,经PEG-400分离纯化,比活性为6.1 U/mg,纯化倍数为4倍。WcCab3-rDSR表达为可溶性均质蛋白,SDS-PAGE分析分子量约为180 kDa。在35℃、pH 5.0、50 mM醋酸钠缓冲液中酶活性最高。WcCab3-rDSR的Km为6.2 mM, Vm为6.3µmol/min/mg。对WcCab3-rDSR合成的葡聚糖进行了傅里叶变换红外和核磁共振波谱分析,发现其结构与天然葡聚糖蔗糖酶合成的葡聚糖具有相似性。以reuteri乳杆菌(protein database, PDB id: 3HZ3)和变形链球菌(PDB id: 3AIB)的葡聚糖酶晶体结构为模板,构建WcCab3-rDSR的模型结构,描述了A、B、C、IV和v等不同结构域的存在。结构域A和B在自然界中呈环状排列,具有(β/α)8磷酸三糖异构酶桶状折叠,构成WcCab3-rDSR的催化核心。WcCab3-rDSR与70 GH家族现有酶结构的结构叠加和多序列比对分析表明,氨基酸残基Asp510作为亲核试剂,Glu548作为催化酸/碱,Asp621作为过渡状态稳定剂,这些残基在该家族中被发现是保守的。
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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