BACKGROUND Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (K M 0.06 mM at pH 5), high catalytic efficiencies (1.3 • 10(6) M(-1) • s(-1) at pH 5), pHopt of 5.5 and Topt at 45°C. The enzyme is not thermostable (T½ of 18 minutes at 60°C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 Å for Cα when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.

中文翻译：

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重氮糖杆菌属丙酮酸丙酮酸脱羧酶的结构和功能表征。

背景技术细菌丙酮酸脱羧酶（PDC）是罕见的。然而，它们在乙醇生产和细菌介导的乙醇生成过程中的作用确保了人们持续不断的兴趣。来自运动发酵单胞菌（ZmPDC），棕榈芽孢杆菌（ZpPDC）和室尾藻（Sarcina ventriculi）（SvPDC）的PDC已被表征，ZmPDC已在一系列异源宿主中成功生产。来自醋杆菌科的PDC及其在代谢中的作用尚未得到相同程度的表征。例子包括氧化葡糖杆菌（GoPDC），重氮芽孢杆菌（GdPDC）和巴斯德醋杆菌（ApPDC）。所有这些生物都具有商业重要性。结果本研究报告了重氮糖尿杆菌（GdPDC）的PDC的动力学特征和晶体结构。酶动力学分析表明对丙酮酸有很高的亲和力（pH为5时，KM为0.06 mM），催化效率高（pH为5时为1.3•10（6）M（-1）•s（-1）），pHopt为5.5和Topt在45℃。该酶不是热稳定的（在60°C下，T½为18分钟），单体和二聚体之间的计算键数与其他PDC相比，没有相对较低的热稳定性的明确迹象。比较GdPDC和Apd​​DC时，其结构与运动发酵单胞菌（ZmPDC）和巴氏曲霉PDC（ApPDC）的结构高度相似，Cα的均方根值为0.57Å。吲哚-3-丙酮酸酯不用作酶的底物。在两个基因座中发生结构差异，涉及区域Thr341至Thr352和Asn499至Asp503。结论这是来自G的PDC的首次研究。重氮菌（PAL5），并为进一步研究其在这种共生共生体内的作用奠定了基础。GdPDC的晶体结构表明该酶与运动发酵单胞菌和巴氏曲霉的同源物在进化上密切相关，并显示出强大的选择性压力，可将酶的特性保持在狭窄的范围内。最适的pH值和降低的热稳定性可能反映了宿主生物的生态位和自然选择这些特性的条件。对吲哚-3-丙酮酸酯的缺乏活性排除了该脱羧酶，因为该酶负责重氮营养菌中吲哚乙酸的产生。巴氏杆菌，并建议强大的选择压力，以保持酶特性在狭窄的范围内。最适的pH值和降低的热稳定性可能反映了宿主生物的生态位和自然选择这些特性的条件。对吲哚-3-丙酮酸酯的缺乏活性排除了该脱羧酶，因为该酶负责重氮营养菌中吲哚乙酸的产生。巴氏杆菌，并建议强大的选择压力，以保持酶特性在狭窄的范围内。最适的pH值和降低的热稳定性可能反映了宿主生物的生态位和自然选择这些特性的条件。对吲哚-3-丙酮酸酯的缺乏活性排除了该脱羧酶，因为该酶负责重氮营养菌中吲哚乙酸的产生。