Simultaneous Involvement of a Tungsten-Containing Aldehyde: Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism
Simultaneous Involvement of a Tungsten-Containing Aldehyde: Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism
复制标题
DOI：
10.1128/jb.00980-13
复制
发表时间：
2014-01-01
影响因子：
3.2
通讯作者：
Heider, Johann
中科院分区：
文献类型：
--
作者：
Debnar-Daumler, Carlotta;Seubert, Andreas;Heider, Johann
厌氧苯丙氨酸代谢的β-变形杆菌芳香芳香族的起始苯丙氨酸转化为苯乙酸，这是进一步代谢通过苯甲酰辅酶A（CoA）。苯乙酸的形成由苯丙氨酸转氨酶、苯丙酮酸脱羧酶和苯丙酮酸脱氧酶催化。在苯丙氨酸和硝酸盐生长的细胞提取物中检测到这些酶的存在。我们发现，两种不同的酶参与苯乙醛氧化为苯乙酸，醛：铁氧还蛋白氧化还原酶（AOR）和苯乙醛脱氢酶（PDH）。基于序列比较，不同钨酸盐浓度的生长研究，以及富集酶的金属分析，AOR被证明是一种含钨的酶，需要特定的辅因子生物合成途径，同时为钨和钨依赖性酶。我们预测，从基因组序列，大多数酶的多蝶呤生物合成的共享，而重复和专门的旁系同源的硫插入MoaD和金属插入MoeA蛋白质似乎参与专门的生物合成对钼或钨辅因子。我们还对PDH进行了生物化学表征，并确定NAD（+）和NADP（+）都是电子受体。我们鉴定了编码该酶的基因，并纯化了重组Streptaged PDH变体。同源四聚体酶对苯乙醛具有高度特异性，对底物具有协同动力学，并显示出相当大的底物抑制作用。我们的数据表明，A.芳香酸利用PDH作为厌氧苯丙氨酸降解过程中的主要酶，而AOR不是代谢途径所必需的。我们假设一个功能作为解毒酶，如果高醛浓度积累在细胞质中，这将导致底物抑制PDH。
Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde: ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum-and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Streptagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.