Ohta Y, Higgins I, Ribbons D W
J Biol Chem. 1975 May 25;250(10):3814-25.
Orcinol hydroxylase (EC 1.14.13.6), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized. Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone. The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm. FAD can be dissociated from the protein. Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN. The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein. K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY. The molecular activity of the crystalline enzyme is 1560 min minus 1. In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2. Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol. Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated. The latter compounds are not hydroxylated. Instead H2O2 accumulates as the only product of O2 reduction. The enzyme therefore behaves either as a hydroxylase or an oxidase. The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation. The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol. K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy. The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol. The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol. Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector. Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur. Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector. The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation.
苔黑酚羟化酶（EC 1.14.13.6）催化恶臭假单胞菌01中苔黑酚分解代谢的第一步反应，已被纯化至同质并结晶。苔黑酚羟化酶催化苔黑酚的羟基化反应，消耗等摩尔的O₂和NADH（或NADPH）生成2,3,5 - 三羟基甲苯，后者非酶氧化为醌。该酶的可见吸收光谱在373和454 nm处有最大值，在480 nm处有一个肩峰。FAD可从蛋白质上解离。用FAD可实现酶活性的重建，FMN在一定程度上也能实现。该酶的分子量为63,000至68,000，每摩尔蛋白质含有1摩尔FAD。苔黑酚、NADH和O₂这三种底物的K-m值分别为0.03、0.13和0.07 mM。结晶酶的分子活性为1560 min⁻¹。在没有苔黑酚的情况下，NADH仅缓慢氧化生成H₂O₂。苔黑酚的几种类似物也可作为羟基化的底物，即间苯二酚、4 - 甲基间苯二酚和4 - 溴间苯二酚。其他类似物，如间甲酚、间乙苯酚、4 - 乙基间苯二酚和间苯三酚，作为效应物模拟苔黑酚，因为它们（a）加速电子从NADH流向黄素，（b）降低NADH的表观K-m值，但程度不如被羟基化的底物。后一类化合物不被羟基化。相反，H₂O₂作为O₂还原的唯一产物积累。因此，该酶表现为羟化酶或氧化酶。随着孵育温度升高，该酶的羟化酶与氧化酶活性之比降低；在60℃时，与苔黑酚的反应几乎有50%与羟基化解偶联。效应物的表观K-m值与苔黑酚、间苯二酚和间甲酚获得的D-D值高度一致。通过差示吸收光谱法测量效应物诱导的黄素蛋白可见吸收光谱的扰动来获得K-D值。在苔黑酚存在下，苔黑酚羟化酶的圆二色光谱也会改变。黄素在整个反应中的参与通过在厌氧条件下，在苔黑酚、间苯二酚或间甲酚存在时NADH对其的快速还原得以证明。随后引入氧气可恢复氧化形式，当间甲酚作为效应物时会产生H₂O₂，但当苔黑酚作为效应物时则不会。还原当量也可能从还原的黄素蛋白转移至游离FAD。在没有效应物的情况下，NADH对苔黑酚羟化酶的还原速度比在有效应物存在时慢10⁻⁴倍。效应物的最小结构要求似乎是1,3 - 二羟基或1 - 烷基 - 3 - 羟基苯，但只有前者是羟基化的底物。
