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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Biochemical characterization of TyrA enzymes from Ignicoccus hospitalis and Haemophilus influenzae: A comparative study of the bifunctional and monofunctional dehydrogenase forms
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Biochemical characterization of TyrA enzymes from Ignicoccus hospitalis and Haemophilus influenzae: A comparative study of the bifunctional and monofunctional dehydrogenase forms

机译：Ignicoccus Headyis和Haemophilus流感的噻嗪酶的生化特征：双功能和单官能脱氢酶形式的比较研究

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摘要

Biosynthesis L-tyrosine (L-Tyr) is directed by the interplay of two enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which is then converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD). This work reports the first characterization of the independently expressed PD domain of bifunctional CM-PD from the crenarchaeon Ignicoccus hospitalis and the first functional studies of both full-length CM-PD and the PD domain from the bacterium Haemophilus influenzae. All proteins were hexa-histidine tagged, expressed in Escherichia coli and purified. Expression and purification of I. hospitalis CM-PD generated a degradation product identified as a PD fragment lacking the protein's first 80 residues, Delta 80CM-PD. A comparable stable PD domain could also be generated by limited Cryptic digestion of this bifunctional enzyme. Thus, Delta 80CM-PD constructs were prepared in both organisms. CM-PD and Delta 80CM-PD from both organisms were dimeric and displayed the predicted enzymatic activities and thermal stabilities in accord with their hyperthermophilic and mesophilic origins. In contrast with H. influenzae PD activity which was NAD(+)-specific and displayed >75% inhibition with 50 mu M L-Tyr, I. hospitalis PD demonstrated dual cofactor specificity with a preference for NADP+ and an insensitivity to L-Tyr. These properties are consistent with a model of the I. hospitalis PD domain based on the previously reported structure of the H. influenzae homolog. Our results highlight the similarities and differences between the archaeal and bacterial TyrA proteins and reveal that the PD activity of both prokaryotes can be successfully mapped to a functionally independent unit. (C) 2016 Elsevier B.V. All rights reserved.
机译：Biosynthesis L-酪氨酸（L-Tyr）由两种酶的相互作用引导。酸毒水解变异（cm）催化酸素对哌妥酸盐的重排，然后通过哌膦酸脱氢酶（Pd）转化为羟基苯基吡酰磺酸盐。该工作报告了从Crenarchaeon Ignicoccus Headyis的独立表达的双功能CM-PD的第一次表达的双官能CM-PD和来自嗜血杆菌嗜血杆菌的全长CM-Pd和PD结构域的第一功能研究。所有蛋白质均为六粒组氨酸标记，在大肠杆菌中表达并纯化。 I. Heady Heady is Cm-Pd的表达和纯化产生了鉴定为缺乏蛋白质的前80个残基的Pd片段的降解产物，Delta 80cm-Pd。也可以通过该双官能酶的有限密码消化产生可比较的稳定PD结构域。因此，在两种生物中制备了Delta 80CM-PD构建体。来自两种生物的CM-Pd和Delta 80cm-Pd是二聚体的，并以符合其高热和嗜苯胺的起源呈现预测的酶活性和热稳定性。与H.Compenenza PD活性相比，NAD（+） - 特异性和显示> 75％抑制50 mu m L-Tyr，I. Heady Headys PD展示了双辅因子特异性，偏好于NADP +和对L-TYR的不敏感性。这些性质与I. Headysis Pd结构域的模型一致，基于先前报道的H.Compenzae同源物的结构。我们的结果突出了古物和细菌泰国蛋白之间的相似性和差异，并表明两种原核生物的PD活性可以成功映射到功能独立的单位。 （c）2016年Elsevier B.v.保留所有权利。

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