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首页> 外文期刊>Biochemistry >Characterization of Soluble and Membrane-Bound Family 3 Lytic Transglycosylases from Pseudomonas aeruginosa
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Characterization of Soluble and Membrane-Bound Family 3 Lytic Transglycosylases from Pseudomonas aeruginosa

机译：铜绿假单胞菌的可溶性和膜绑定的家庭3裂解转糖基酶的表征。

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摘要

Lytic transglycosylases cleave the #beta#1->4 glycosidic linkages between the N-acetylmuramoyl (MurNAc) and N-acetylglucosaminyl (GlcNAc) residues of peptidoglycan with the concomitant formation of 1,6-anhydro-N-acetylmuramyl reaction products. The genes encoding two hypothetical lytic transgly-cosylases were identified in the genome of Pseudomonas aeruginosa PAO1 by a BLAST search using membrane-bound lytic transglycosylase B (MltB) from Escherichia coli as the query. The two genes were amplified by PCR and cloned as fusion proteins with C-terminal hexa-His sequences. Expression studies of the two genes in E. coli in the presence of [~3H]palmitate resulted in the labeling of only one of the two enzymes. This enzyme, named MltB, was overexpressed to form insoluble inclusion bodies. Its gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which includes Cys17, the putative site of lipidation. This MltB derivative (named sMltB) was shown to not label with [~3H]palmitate, and it was overexpressed in soluble form. The second, nonlabeled enzyme was overexpressed in soluble form and hence was named soluble lytic transglycosylase B (SltB). Both sMltB and SltB were purified to apparent homogeneith by a combination of affinity (Ni~(2+)-NTA), cation-exchange (Mono S), and gel permeation (Superdex 75) chromatographies. The reaction products released by the two enzymes from purified, insoluble peptidoglycan were characterized by a novel high-performance anion-exchange chromatography (HPAEC) assay. Both enzymes produced the same three major soluble products which were identified as anhydromuropeptides based on ESI-MS analysis (cross-linked anhydrodisaccharide-tetrasaccharide, m/z obs 1824.9; anhydrodisaccharide-pentapeptide, m/z obs 922.2; and anhydrodisaccharide-tripeptide, m/z obs 851.3. The Michaelis-Menten kinetic parameters were also determined for the two enzymes using the same insoluble peptidoglycan substrate by aminosugar compositional analysis of soluble reaction products. At pH 5.8 and in the presence of 0.1% Triton, SltB was found to be more catalycally efficient, as reflected by its k_(cat)/K_M value, than sMltB.
机译：溶血性糖基转移酶切割肽聚糖的N-乙酰基村酰（MurNAc）和N-乙酰基氨基葡萄糖基（GlcNAc）残基之间的＃β＃1-4糖苷键，并伴随形成1,6-脱水-N-乙酰基村m基反应产物。铜绿假单胞菌PAO1的基因组中，通过使用来自大肠杆菌的膜结合裂解转糖基化酶B（MltB）进行BLAST搜索，鉴定出编码两个假设的裂解转糖基化酶的基因。通过PCR扩增这两个基因，并将其克隆为具有C末端hexa-His序列的融合蛋白。在[〜3H]棕榈酸存在下，两种基因在大肠杆菌中的表达研究导致仅标记了两种酶中的一种。该酶名为MltB，过表达形成不溶性包涵体。其基因被工程化以产生缺少其N-末端17个残基的酶的截短形式，该基因被工程化以产生缺乏其N-末端17个残基的酶的截短形式，包括Cys17，假定的脂质化位点。该MltB衍生物（名为sMltB）显示未用[〜3H]棕榈酸酯标记，并且以可溶形式过表达。第二种未标记的酶以可溶形式过度表达，因此被称为可溶性裂解转糖基酶B（SltB）。 sMltB和SltB均通过亲和力（Ni〜（2 +）-NTA），阳离子交换（Mono S）和凝胶渗透（Superdex 75）色谱法组合纯化为表观同质。两种酶从纯化的不溶性肽聚糖中释放的反应产物通过新型高效阴离子交换色谱（HPAEC）分析进行了表征。两种酶均产生相同的三种主要可溶性产物，根据ESI-MS分析将其鉴定为脱水多肽（交联脱水二糖-四糖，m / z obs 1824.9;脱水二糖-五肽，m / z obs 922.2;和脱水二糖-三肽，m / z obs 851.3。使用相同的不溶性肽聚糖底物，通过可溶性反应产物的氨基糖组成分析，还确定了两种酶的Michaelis-Menten动力学参数，在pH 5.8和存在0.1％Triton的情况下，发现SltB为由其k_（cat）/ K_M值反映，比sMltB具有更高的催化效率。

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