Characterization of the β-methylaspartate-α-decarboxylase (CrpG) from the cryptophycin Biosynthetic pathway
Characterization of the β-methylaspartate-α-decarboxylase (CrpG) from the cryptophycin Biosynthetic pathway
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DOI：
10.1002/cbic.200700162
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发表时间：
2007-08-13
期刊：
影响因子：
3.2
通讯作者：
Sherman, David H.
中科院分区：
文献类型：
--
作者：
Beck, Zachary Q.;Burr, Douglas A.;Sherman, David H.
念珠藻素是有前途的治疗剂，由于其对癌细胞的有效选择性，包括多药耐药肿瘤细胞系，以及它们逃避p-糖蛋白泵的能力。[1，2]最近在念珠藻属ATCC 53789和念珠藻属GSV 224中鉴定了负责产生念珠藻素的生物合成簇。[3]未来合理设计新型念珠藻素类似物生物合成的努力依赖于对参与构建这些有价值的天然产物的每种相应酶的作用的详细理解。超过25种天然存在的念珠藻素中的大多数由四个亚基组成：单元A，苯基辛酸，单元B，3-氯-O-甲基-D-酪氨酸，单元C，3-氨基-2（R）-甲基丙-ACHTUNGTRENNUNG酸，和单元D，L-亮氨酸（方案1）。前体掺入研究表明，作为单元C整合到念珠藻素中的3-氨基-2（R）-甲基丙酸酯是通过（2S，3R）-3-甲基天冬氨酸（1）脱羧产生的。[3]然而，尚未有关于在微生物初级或次级代谢中具有经证实的β-甲基天冬氨酸-α-脱羧酶活性的酶的报道。此外，至少五种念珠藻素类似物含有β-丙氨酸作为单元C。对念珠藻素生物合成基因簇[3]的生物信息学分析显示，它含有一个蛋白产物（CrpG）的开放阅读框，该蛋白产物与异戊酰依赖性天冬氨酸脱羧酶具有高度相似性（图1）。天冬酰依赖性天冬氨酸脱羧酶是一组独特的机械相关酶的成员，包括S-腺苷甲硫氨酸脱羧酶、磷脂酰丝氨酸脱羧酶、脯氨酸还原酶和细菌组氨酸脱羧酶。这些酶最初以酶原形式（π）表达，其通过内部丝氨酸残基在XnisSer键处被蛋白水解切割，以产生在其N末端含有异戊酰基的β亚基和含有C末端酸的α亚基（方案2A）。[4]天冬氨酸脱羧酶（PanD）是天冬氨酸脱羧酶的一个重要组成部分，它与氨基酸的胺形成Schiff碱（反应图2 B）。大肠杆菌不能结合β-甲基天冬氨酸的所有可能的立体异构体。[5]此外，还对E.大肠杆菌和幽门螺杆菌L-天冬氨酸脱羧酶在其各自的活性位点中显示出高度保守的氨基酸，这些活性位点与底物直接接触。[6，7]这些氨基酸残基与CrpG的同一性比较表明，以下三个氨基酸残基在CrpG中是变体：Gly 72、Ser 73和Thr 84（图1）。该ACHTUNGTRENNUNG信息，结合结合crpG掺入研究结果，使我们假设假定假定的crpG脱羧酶基因产物负责1的脱羧形成3-氨基-2（R）-甲基丙酸，其随后掺入念珠藻素中（方案1）。在这份报告中，我们描述了克隆，基因过表达，纯化的crpG基因产物，合成的底物，和生化特性的CrpG底物特异性。最初对念珠藻素crpG表达载体进行了工程改造，以产生带有C末端His 6标签的CrpG，因为这种类型的翻译融合蛋白不会干扰来自H的天冬氨酸脱羧酶的功能。幽门。[8]然而，从该构建体获得的蛋白质的低酶活性表明His 6-标签确实显著损害了CrpG功能。
The cryptophycins are promising therapeutic agents due to their potent selectivity for cancer cells, which include multidrug-resistant tumor cell lines, and their ability to evade p-glycoprotein pumps.[1, 2] The biosynthetic cluster responsible for production of the cryptophycins has been identified recently in Nostoc sp. ATCC53789 and Nostoc sp. GSV224.[3] Future ACHTUNGTRENNUNGefforts to rationally engineer the biosynthesis of novel cryptophycin analogues relies on a detailed understanding of the role for each of the corresponding enzymes involved in construction of these valuable natural products. The majority of the more than 25 naturally occurring cryptophycins are composed of four subunits: unit A, phenyloctanoic acid, unit B, 3-chloro-O-methyl-D-tyrosine, unitC, 3-amino-2 (R)-methylpropi-ACHTUNGTRENNUNGonic acid, and unit D, L-leucic acid (Scheme 1). Precursor-incorporation studies have indicated that 3-amino-2 (R)-methylpropionate integrated as unit C into the cryptophycins is generated by decarboxylation of (2S, 3R)-3-methylaspartate (1).[3] However, there have been no reports of an enzyme with confirmed β-methylaspartate-α-decarboxylase activity in microbial primary or secondary metabolism. In addition, at least five cryptophycin analogues contain β-alanine as unit C. Bioinformatic analysis of the cryptophycin biosynthetic gene cluster [3] has revealed that it contains an open-reading frame for a protein product (CrpG) that bears high similarity to pyruvoyl-dependent aspartate decarboxylases (Figure 1). The pyruvoyl-dependent aspartate decarboxylases are members of a unique group of mechanistically related enzymes, which include S-adenosylmethionine decarboxylase, phosphatidylserine decarboxylase, proline reductase, and bacterial histidine decarboxylase. These enzymes are initially expressed in a proenzyme form (π), which is proteolytically cleaved at an XÀ Ser bond by an internal serine residue, to produce a β-subunit that contains a pyruvoyl group at its N terminus and an αsubunit that contains a C-terminal acid (Scheme 2A).[4] As a ACHTUNGTRENNUNGprelude to catalysis, the pyruvoyl group forms a Schiff’s base with the amine of the amino acid to be decarboxylated (Scheme 2 B).Previous studies have shown that the aspartate decarboxylase (PanD) from E. coli fails to bind all possible stereoisomers of β-methylaspartate.[5] Moreover, analysis of the crystal structures of E. coli and Helicobacter pylori L-aspartate decarboxylases revealed highly conserved amino acids in their respective active sites that are in direct contact with the substrate.[6, 7] Comparison of the identity of these amino acid residues with CrpG indicated that the following three amino acid residues are variant in CrpG: Gly72, Ser73, and Thr84 (Figure 1). This ACHTUNGTRENNUNGinformation, in conjunction with the precursor-incorporation study results, led us to hypothesize that the putative crpG decarboxylase gene product is responsible for the decarboxylation of 1 to form 3-amino-2 (R)-methylpropionic acid, which is subsequently incorporated into the cryptophycins (Scheme 1). In this report we describe the cloning, gene over-expression, purification of the crpG gene product, synthesis of substrates, and biochemical characterization of CrpG substrate specificity. The cryptophycin crpG expression vector was initially engineered to produce CrpG with a C-terminal His6-tag, as this type of translational fusion protein did not interfere with the function of aspartate decarboxylase from H. pylori.[8] However, the low enzymatic activity of the protein obtained from this construct indicated that the His6-tag did significantly compromise CrpG function …