Identification in Agrobacterium tumefaciens of the d-galacturonic acid dehydrogenase gene
Identification in Agrobacterium tumefaciens of the d-galacturonic acid dehydrogenase gene
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DOI：
10.1007/s00253-009-2333-9
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发表时间：
2010-04-01
影响因子：
5
通讯作者：
Richard, Peter
中科院分区：
文献类型：
--
作者：
Boer, Harry;Maaheimo, Hannu;Richard, Peter
微生物中d-半乳糖醛酸的催化至少有三种不同的途径。在一些原核生物物种中描述的氧化途径中，d-半乳糖醛酸首先被烟酰胺腺嘌呤二核苷酸（NAD）依赖性脱氢酶（EC 1.1.1.203）氧化为内消旋半乳糖二酸（粘酸）。在该途径的以下步骤中，粘酸盐转化为2-酮戊二酸盐。这种分解代谢途径的酶活性已被描述，而相应的基因序列仍然是未知的。从根癌农杆菌中纯化了d-半乳糖醛酸脱氢酶，并使用MALDI-TOF质谱法测定了其胰蛋白酶肽的质量。这使得能够识别相应的基因udh。它编码具有267个氨基酸的蛋白质，该蛋白质与NAD（P）结合罗斯曼折叠蛋白的蛋白质家族具有同源性。开放阅读框在酿酒酵母中得到功能性表达。N-末端标记的蛋白质在其活性上没有受损，并且在纯化后用于动力学表征。该酶对NAD具有特异性，并接受D-半乳糖醛酸和D-葡萄糖醛酸作为具有相似亲和力的底物。NMR分析表明，在水溶液中，底物d-半乳糖醛酸主要以吡喃糖形式存在，吡喃糖形式被酶转化为半乳糖醛酸的1，4内酯。这种内酯在细胞内条件下似乎是稳定的，并且不会自发地打开线性内消旋半乳糖苷酸。
There are at least three different pathways for the catabolism of d-galacturonate in microorganisms. In the oxidative pathway, which was described in some prokaryotic species, d-galacturonate is first oxidised to meso-galactarate (mucate) by a nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase (EC 1.1.1.203). In the following steps of the pathway mucate is converted to 2-keto-glutarate. The enzyme activities of this catabolic pathway have been described while the corresponding gene sequences are still unidentified. The d-galacturonate dehydrogenase was purified from Agrobacterium tumefaciens, and the mass of its tryptic peptides was determined using MALDI-TOF mass spectrometry. This enabled the identification of the corresponding gene udh. It codes for a protein with 267 amino acids having homology to the protein family of NAD(P)-binding Rossmann-fold proteins. The open reading frame was functionally expressed in Saccharomyces cerevisiae. The N-terminally tagged protein was not compromised in its activity and was used after purification for a kinetic characterization. The enzyme was specific for NAD and accepted d-galacturonic acid and d-glucuronic acid as substrates with similar affinities. NMR analysis showed that in water solution the substrate d-galacturonic acid is predominantly in pyranosic form which is converted by the enzyme to 1,4 lactone of galactaric acid. This lactone seems stable under intracellular conditions and does not spontaneously open to the linear meso-galactaric acid.