Pyrroloquinoline quinone biogenesis: Characterization of PqqC and its H84N and H84A active site variants
Pyrroloquinoline quinone biogenesis: Characterization of PqqC and its H84N and H84A active site variants
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DOI：
10.1021/bi700162n
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发表时间：
2007-06-19
期刊：
影响因子：
2.9
通讯作者：
Klinman, Judith P.
中科院分区：
文献类型：
--
作者：
Magnusson, Olafur Th.;RoseFigura, Jordan M.;Klinman, Judith P.
吡咯喹啉醌[4，5-dihydro-4，5-dioxo-1H-pyrrolo[2，3-f]quinoline-2，7，9-tricarboxylic酸(PQQ)]是一种细菌维生素，在许多酒精脱氢酶中充当辅因子。它在肺炎克雷伯菌中的生物合成受到pqqABCDEF六个基因的促进，并通过一条未知的途径进行。PqqC编码的蛋白质催化PQQ形成的最后一步，在没有氧化还原活性金属或辅因子的情况下，这一步骤包括环的闭合和3a-(2-amino-2-carboxyethyl)-4，5-dioxo-4，5，6，7，8，9-hexahydroquinoline-7，9-dicarboxylic酸的整体八电子氧化。最近的晶体结构涉及到许多PQQ-PqQC相互作用[Magnusson等人。(2004)Proc.娜塔莉。阿卡德。SCI。美国第101号，7913-7918号]。为了研究PqqC反应的机理，将活性中心残基His84突变为H84A和H84N，并在有氧和厌氧条件下比较了两者和野生型酶的动力学和光谱性质。这两个突变体在有氧条件下形成PQQ，野生型酶的速率常数分别为0.09min(-1)和0.056 min(-1)，而野生型酶的速率常数为0.34min(-1)。除了最初的E-AHQQ复合体(532-536 nm)和产物E-PQQ复合体(346-366 nm)外，在316-344 nm之间还观察到一些光谱中间体。厌氧反应特别有意义，表明虽然H84N与AHQQ的混合产生了344 nm的中间体，但这不能进行到最终的318 nm物种；相比之下，H84A形成了344 nm物种作为318 nm物种的前体。在PqqC的拟议化学机理方面[Magnusson等人。(2004)Proc.娜塔莉。阿卡德。SCI。U.S.A.101,7913-7918]，我们将344 nm的中间体指定为喹类化合物物种，并将318 nm的中间体指定为初始的喹酚物种。所提出的H84的作用是作为喹类物种的氧阴离子的质子供体，以便随后发生C-H键断裂以形成单阴离子的喹酚。在没有质子给体的情况下(如在H84N中发生的情况)，正常的反应路径被排除，因为这将需要形成不稳定的双阴离子物种。与H84N不同，H84A看起来足够小，可以允许活性中心水进入，这被认为是活性中心质子供体的作用。
Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial vitamin that serves as a cofactor in numerous alcohol dehydrogenases. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF, and proceeds by an unknown pathway. The protein encoded by pqqC catalyzes the final step of PQQ formation, which involves a ring closure and an overall eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) in the absence of a redox-active metal or cofactor. A recent crystal structure has implicated numerous PQQ-PqqC interactions [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918]. To investigate the mechanism of the PqqC reaction, the active site residue His84 has been mutated to H84A and H84N, and the kinetic and spectroscopic properties have been compared to each other and the wild-type enzyme using aerobic and anaerobic conditions. Both mutants form PQQ under aerobic conditions with rate constants of 0.09 min(-1) and 0.056 min(-1) relative to 0.34 min(-1) for the wild-type enzyme. In addition to the initial E-AHQQ complex (532-536 nm) and the product E-PQQ complex (346-366 nm), a number of spectral intermediates are observed between 316 and 344 nm. The anaerobic reaction is particularly informative, showing that while mixing of H84N with AHQQ leads to a 344 nm intermediate, this is unable to proceed to a final 318 nm species; by contrast H84A forms the 344 nm species as a precursor to the 318 nm species. In the context of the proposed chemical mechanism for PqqC [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918], we assign the 344 nm intermediate to a quinoid species and the 318 nm intermediate to an initial quinol species. The proposed role of H84 is as a proton donor to the oxyanion of the quinoid species such that subsequent C-H bond cleavage can occur to form a monoanionic quinol. In the absence of a proton donor (as occurs in H84N), the normal reaction path is precluded as this would require formation of an unstable, dianionic species. Unlike H84N, H84A appears to be small enough to allow entry of active site water, which is postulated to adopt the role of active site proton donor.