Concerted action of diacetylchitobiose deacetylase and exo-β-D-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
Concerted action of diacetylchitobiose deacetylase and exo-β-D-glucosaminidase in a novel chitinolytic pathway in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
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DOI：
10.1074/jbc.m314187200
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发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Imanaka, T
中科院分区：
文献类型：
--
作者：
Tanaka, T;Fukui, T;Imanaka, T
极端嗜热古菌Thermococcus kodakaraensis KOD 1具有几丁质酶（Tk-ChiA）和外切-β-D-氨基葡萄糖苷酶（Tk-GlmA）降解几丁质;前者从几丁质产生二乙酰壳二糖（GlcNAc（2）），后者将壳二糖（GlcN（2））水解为葡萄糖胺（GlcN）。为了鉴定生理上连接这两种活性的酶，在这里我们集中于为来自GlcNAc 2的Tk-GlmA提供底物的脱乙酰酶。从T. kodakaraensis细胞，并在基因组上鉴定了相应的基因（Tk-fos）。推导的氨基酸序列属于LmbE蛋白家族，包括N-乙酰氨基葡萄糖磷脂酰肌醇脱N-乙酰酶和1-D-肌醇-2-乙酰氨基-2-脱氧-α-D-吡喃葡萄糖苷脱乙酰酶。重组Tk-Dac对N-乙酰基壳寡糖（GlcNAc（2-5））具有脱乙酰酶活性，且脱乙酰化位点特异于非还原性GlcNAc残基。该酶还使GlcNAc单体脱乙酰化。于T.在kodakaraensis细胞中，GlcNAc可诱导Tk-glmA、Tk-chiA和Tk-chiA基因簇的转录（2），提示该基因簇在体内几丁质酶催化中的功能。这些结果揭示了T. Kodakaraensis中几丁质产生的GlcNAc（2）在Tk-Dac和Tk-GlmA的协同作用下被降解。也就是说，GlcNAc（2）通过Tk-Dac位点特异性地脱乙酰化为GlcN-GlcNAc，然后通过Tk-GlmA水解为GlcN和GlcNAc，随后通过Tk-Dac对剩余的GlcNAc进行第二脱乙酰化步骤以形成GlcN。这是第一次阐明古细菌几丁质分解代谢途径，并定义了一种新的机制，二聚体加工使用的组合脱乙酰基和裂解，不同于任何以前已知的途径。
The hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 possesses chitinase (Tk-ChiA) and exo-beta-D-glucosaminidase (Tk-GlmA) for chitin degradation; the former produces diacetylchitobiose (GlcNAc(2)) from chitin, and the latter hydrolyzes chitobiose (GlcN(2)) to glucosamine ( GlcN). To identify the enzyme that physiologically links these two activities, here we focused on the deacetylase that provides the substrate for Tk-GlmA from GlcNAc2. The deacetylase could be detected in and partially purified from T. kodakaraensis cells, and the corresponding gene (Tk-dac) was identified on the genome. The deduced amino acid sequence was classified into the LmbE protein family including N-acetylglucosaminylphosphatidylinositol de-N-acetylases and 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase. Recombinant Tk-Dac showed deacetylase activity toward N-acetylchitooligosaccharides (GlcNAc(2-5)), and the deacetylation site was revealed to be specific at the nonreducing GlcNAc residue. The enzyme also deacetylated GlcNAc monomer. In T. kodakaraensis cells, the transcription of Tk-dac, Tk-glmA, Tk-chiA, and the clustered genes were induced by GlcNAc(2), suggesting the function of this gene cluster in chitin catabolism in vivo. These results have revealed a unique chitin catabolic pathway in T. kodakaraensis, in which GlcNAc(2) produced from chitin is degraded by the concerted action of Tk-Dac and Tk-GlmA. That is, GlcNAc(2) is site-specifically deacetylated to GlcN-GlcNAc by Tk-Dac and then hydrolyzed to GlcN and GlcNAc by Tk-GlmA followed by a second deacetylation step of the remaining GlcNAc by Tk-Dac to form GlcN. This is the first elucidation of an archaeal chitin catabolic pathway and defines a novel mechanism for dimer processing using a combination of deacetylation and cleavage, distinct from any previously known pathway.