Arias-Barrau Elsa, Sandoval Angel, Naharro Germán, Olivera Elías R, Luengo José M
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, 24007 León, Spain.
J Biol Chem. 2005 Jul 15;280(28):26435-47. doi: 10.1074/jbc.M501988200. Epub 2005 May 2.
The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, l-Phe, and l-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).
已确定恶臭假单胞菌U中参与3-羟基苯乙酸（3-OH-PhAc）同化作用的完整分解代谢途径。该途径由以下部分组成：（i）一条特定途径（上游途径），催化3-OH-PhAc转化为2,5-二羟基苯乙酸（2,5-二OH-PhAc，即尿黑酸，Hmg）；（ii）一条中心途径（汇聚途径），催化由3-OH-PhAc、L-苯丙氨酸（L-Phe）和L-酪氨酸（L-Tyr）生成的Hmg转化为富马酸和乙酰乙酸（HmgABC）。因此，在第一步中，3-OH-PhAc的降解需要通过一个主动运输系统摄取3-OH-PhAc，该系统涉及一种通透酶（MhaC）的参与以及磷酸烯醇丙酮酸作为能量来源。一旦3-OH-PhAc被摄取，它会通过一种新型的双组分黄素蛋白芳香羟化酶（MhaAB）催化的酶促反应被羟基化为2,5-二OH-PhAc。大组分（MhaA，62719 Da）是一种黄素蛋白，小组分（MhaB，6348 Da）是一种偶联蛋白，对于3-OH-PhAc羟基化为2,5-二OH-PhAc至关重要。序列分析和分子生物学研究表明，尿黑酸合酶（MhaAB）与迄今报道的芳香羟化酶不同，这解释了其在3-OH-PhAc分解代谢中的特定作用。此外，还鉴定出了一个参与尿黑酸摄取的ABC转运系统（HmgDEFGHI）和两个调控元件（mhaSR和hmgR）。此外，一些分解代谢基因在不同微生物中的克隆和表达赋予了它们合成Hmg的能力（mhaAB），或者使它们能够在以3-OH-PhAc作为唯一碳源的化学限定培养基中生长（mhaAB和hmgABC）。
