Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases
Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases
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DOI：
10.1016/j.phytochem.2014.07.007
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发表时间：
2014-10-01
期刊：
影响因子：
3.8
通讯作者：
Li, Jianyong
中科院分区：
文献类型：
--
作者：
Torrens-Spence, Michael P.;Lazear, Michael;Li, Jianyong
植物芳香族氨基酸脱羧酶（AAAD）催化芳香族氨基酸与苯环或吲哚环的脱羧反应。由于AAAD的底物选择性与其生理功能密切相关，因此一级序列数据及其差异可以提供重要的生理学见解。然而，由于一般的高序列同一性，植物AAAD底物特异性已经难以通过一级序列比较来鉴定。在这项研究中，生物信息学的方法被用来确定几个活性位点残基内的植物AAAD酶，可能会影响底物特异性。接下来，选择罂粟酪氨酸脱羧酶（TyDC）作为模型，通过突变来验证我们假定的底物决定残基。结果表明，丝氨酸372突变为甘氨酸使得催眠草TyDC能够使用5-羟基色氨酸作为底物，并且降低了对3，4-二羟基-L-苯丙氨酸（多巴）的酶活性。此外，长春花色氨酸脱羧酶（TDC）中的回复突变使得突变酶能够利用酪氨酸和多巴作为底物，对色氨酸的亲和力降低。催眠草TyDC和玫瑰花TDC酶的分子建模和分子对接为解释底物特异性的改变提供了结构基础。影响底物选择性的活性位点残基的鉴定产生了一级序列标识符，其可以帮助区分个别植物AAAD的吲哚和酚底物特异性。(C)2014爱思唯尔有限公司版权所有。
Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs. (C) 2014 Elsevier Ltd. All rights reserved.