Hirsch W, Schägger H, Fuchs G
Mikrobiologie, Institut Biologie II, Universität Freiburg, Germany.
Eur J Biochem. 1998 Feb 1;251(3):907-15. doi: 10.1046/j.1432-1327.1998.2510907.x.
Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.
苯乙酮酸（苯甲酰甲酸）是苯丙氨酸和苯乙酸无氧代谢过程中的一种中间体。它由苯乙酰辅酶A经α-氧化形成。苯乙酮酸被苯乙酮酸氧化还原酶氧化脱羧生成苯甲酰辅酶A，这是厌氧芳香族代谢的核心中间体。在以苯丙氨酸、苯乙酸和苯乙酮酸进行厌氧生长时，反硝化细菌埃文斯氏固氮弧菌中的苯乙酮酸氧化酶活性被诱导，但以苯甲酸进行厌氧生长时则不会。新酶苯乙酮酸：受体氧化还原酶被纯化并进行了研究。这种对氧敏感的酶能还原NAD⁺和紫精染料。它由五个亚基组成，分子量分别约为50、48、43、24和11.5 kDa；通过凝胶过滤测定的天然分子量为370 kDa，表明其组成为α₂β₂γ₂δ₂ε₂。苯乙酮酸：受体氧化还原酶表现出铁硫蛋白特有的紫外/可见光谱，每摩尔酶含有35±4摩尔铁、36±4摩尔酸不稳定硫和1.1±0.2摩尔FAD。该酶对苯乙酮酸（Km为45 μM）和辅酶A（Km为55 μM）具有特异性；2-氧代异戊酸的氧化速率为前者的15%。在37℃、最佳pH为8的条件下，以苄基紫精为底物时的周转数为46 s⁻¹。该酶催化NAD(P)H：紫精染料转氢反应，NAD(H)是更受青睐的辅酶。它还催化CO₂与底物羧基之间的同位素交换。这些数据与以下假设一致。该酶复合物由四个亚基组成的核心酶α₂β₂γ₂δ₂构成，就像古菌的2-氧代酸：铁氧化还原蛋白氧化还原酶那样；这种复合物能够还原紫精染料。全酶还含有一个ε₂亚基，它催化电子从核心复合物中一个类似小铁氧化还原蛋白的亚基转移到NAD⁺；这个亚基还催化转氢酶反应，携带FAD，类似于铁氧化还原蛋白：NAD(P)⁺氧化还原酶。
