Conversion of Mevalonate 3-Kinase into 5-Phosphomevalonate 3-Kinase by Single Amino Acid Mutations
Conversion of Mevalonate 3-Kinase into 5-Phosphomevalonate 3-Kinase by Single Amino Acid Mutations
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DOI：
10.1128/aem.00256-19
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发表时间：
2019-03
影响因子：
4.4
通讯作者：
Kento Motoyama;Fumiaki Sobue;H. Kawaide;T. Yoshimura;H. Hemmi
中科院分区：
文献类型：
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作者：
Kento Motoyama;Fumiaki Sobue;H. Kawaide;T. Yoshimura;H. Hemmi
类异戊二烯是最大的天然化合物家族，包括重要的生物活性分子，如维生素、激素和天然药物。甲羟戊酸途径是代谢工程的目标，因为它为类异戊二烯生物合成提供前体。甲羟戊酸3-激酶是一种参与修饰的甲羟戊酸途径的酶，对有限的嗜热古菌物种具有特异性。在酶的活性位点上替换一个氨基酸残基改变了其底物偏好，并允许突变酶催化以前未发现的反应。利用编码突变体酶和其他古菌酶的基因，我们构建了一个人工甲羟戊酸途径，该途径可以在细菌细胞中通过一个未知的途径产生类异戊二烯的前体。摘要甲羟戊酸3-激酶在最近发现的嗜热古菌特异性的甲羟戊酸途径中起关键作用。该酶与广泛分布的经典甲羟戊酸途径中所涉及的二磷酸甲羟戊酸脱羧酶和嗜盐古菌和某些Chloroflexi细菌所具有的磷酸甲羟戊酸脱羧酶同源。甲羟戊酸3-激酶催化甲羟戊酸的ATP依赖性3-磷酸化，但不像相关脱羧酶那样催化随后的脱羧。在这项研究中，底物相互作用的谷氨酸残基的嗜酸热浆菌甲羟戊酸3-激酶被替换为较小的氨基酸，包括其对应的二磷酸甲羟戊酸脱羧酶和磷酸甲羟戊酸脱羧酶，改变底物特异性的目的。这些单氨基酸突变导致甲羟戊酸3-激酶转化为5-磷酸甲羟戊酸3-激酶，其可以从5-磷酸甲羟戊酸合成3，5-二磷酸甲羟戊酸。催化迄今未发现的反应的突变体使人工甲羟戊酸途径的建设在大肠杆菌细胞中，证明了番茄红素，红色类胡萝卜素色素的积累。重要性类异戊二烯是天然化合物中最大的家族，包括重要的生物活性分子，如维生素、激素和天然药物。甲羟戊酸途径是代谢工程的目标，因为它为类异戊二烯生物合成提供前体。甲羟戊酸3-激酶是一种参与修饰的甲羟戊酸途径的酶，对有限的嗜热古菌物种具有特异性。在酶的活性位点上替换一个氨基酸残基改变了其底物偏好，并允许突变酶催化以前未发现的反应。利用编码突变体酶和其他古菌酶的基因，我们构建了一个人工甲羟戊酸途径，该途径可以在细菌细胞中通过一个未知的途径产生类异戊二烯的前体。
Isoprenoid is the largest family of natural compounds, including important bioactive molecules such as vitamins, hormones, and natural medicines. The mevalonate pathway is a target for metabolic engineering because it supplies precursors for isoprenoid biosynthesis. Mevalonate 3-kinase is an enzyme involved in the modified mevalonate pathway specific to limited species of thermophilic archaea. Replacement of a single amino acid residue in the active site of the enzyme changed its substrate preference and allowed the mutant enzymes to catalyze a previously undiscovered reaction. Using the genes encoding the mutant enzymes and other archaeal enzymes, we constructed an artificial mevalonate pathway, which can produce the precursor of isoprenoid through an unexplored route, in bacterial cells. ABSTRACT Mevalonate 3-kinase plays a key role in a recently discovered modified mevalonate pathway specific to thermophilic archaea of the order Thermoplasmatales. The enzyme is homologous to diphosphomevalonate decarboxylase, which is involved in the widely distributed classical mevalonate pathway, and to phosphomevalonate decarboxylase, which is possessed by halophilic archaea and some Chloroflexi bacteria. Mevalonate 3-kinase catalyzes the ATP-dependent 3-phosphorylation of mevalonate but does not catalyze the subsequent decarboxylation as related decarboxylases do. In this study, a substrate-interacting glutamate residue of Thermoplasma acidophilum mevalonate 3-kinase was replaced by smaller amino acids, including its counterparts in diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, with the aim of altering substrate specificity. These single amino acid mutations resulted in the conversion of mevalonate 3-kinase into 5-phosphomevalonate 3-kinase, which can synthesize 3,5-bisphosphomevalonate from 5-phosphomevalonate. The mutants catalyzing the hitherto undiscovered reaction enabled the construction of an artificial mevalonate pathway in Escherichia coli cells, as was demonstrated by the accumulation of lycopene, a red carotenoid pigment. IMPORTANCE Isoprenoid is the largest family of natural compounds, including important bioactive molecules such as vitamins, hormones, and natural medicines. The mevalonate pathway is a target for metabolic engineering because it supplies precursors for isoprenoid biosynthesis. Mevalonate 3-kinase is an enzyme involved in the modified mevalonate pathway specific to limited species of thermophilic archaea. Replacement of a single amino acid residue in the active site of the enzyme changed its substrate preference and allowed the mutant enzymes to catalyze a previously undiscovered reaction. Using the genes encoding the mutant enzymes and other archaeal enzymes, we constructed an artificial mevalonate pathway, which can produce the precursor of isoprenoid through an unexplored route, in bacterial cells.