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首页> 外文期刊>Journal of Molecular Biology >Crystal structure of E-coli alcohol dehydrogenase YqhD: Evidence of a covalently modified NADP coenzyme
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Crystal structure of E-coli alcohol dehydrogenase YqhD: Evidence of a covalently modified NADP coenzyme

机译：大肠杆菌醇脱氢酶YqhD的晶体结构：共价修饰的NADP辅酶的证据

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In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0 Angstrom resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C-3. YqhD is a dimer of 2 X 387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions. (C) 2004 Elsevier Ltd. All rights reserved.
机译：在旨在解决功能未知的大肠杆菌开放阅读框（ORF）产品结构的结构基因组学计划中，我们使用Pt边缘的单波长异常衍射方法确定了分辨率为2.0埃的YqhD的结构。 YqhD的晶体结构表明它是一种NADP依赖的脱氢酶，通过对几种醇的活性测量证实了这一结果。目前我们对这些发现的解释是，YqhD是一种醇脱氢酶（ADH），优先选择比C-3长的醇。 YqhD是2 X 387个残基的二聚体，每个单体由两个结构域组成，分别是Rossmann型折叠和α螺旋结构域。晶体在不对称单元中包含两个二聚体。虽然二聚体之一在两个亚基中都包含辅因子，但第二二聚体中仅一个亚基包含它，从而可以比较结合和未结合的活性位点。活性位点包含一个Zn原子，通过晶体上的EXAFS验证。 NADP的电子密度图揭示了5和6位上氧原子对烟酰胺环的修饰。进一步的电喷雾质谱分析以及与NADP和NADPH的质谱比较显示了修饰的性质和两个羟基部分的结合在烟酰胺环的5和6位上生成NADPH（OH）（2）。这些修饰可能是由于酶上的氧气压力所致，该酶在厌氧条件下会发挥功能。 （C）2004 Elsevier Ltd.保留所有权利。

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