van Zyl Leonardo J, Schubert Wolf-Dieter, Tuffin Marla I, Cowan Don A
Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Robert Sobukwe Road, Bellville, Cape Town, South Africa.
Department of Biochemistry, University of Pretoria, 2 Lynnwood Road, Pretoria, 0002, South Africa.
BMC Struct Biol. 2014 Nov 5;14:21. doi: 10.1186/s12900-014-0021-1.
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.
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.
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.
细菌丙酮酸脱羧酶（PDC）较为罕见。然而，它们在乙醇生产及细菌介导的乙醇生成过程中的作用，一直引发着持续且不断增长的关注。运动发酵单胞菌（ZmPDC）、棕榈发酵杆菌（ZpPDC）和胃八叠球菌（SvPDC）的PDC已得到表征，并且ZmPDC已在一系列异源宿主中成功表达。醋酸杆菌科的PDC及其在代谢中的作用尚未得到同等程度的表征。例如氧化葡萄糖酸杆菌（GoPDC）、重氮营养葡萄糖酸杆菌（GdPDC）和巴斯德醋酸杆菌（ApPDC）。所有这些生物都具有商业重要性。
本研究报告了重氮营养醋杆菌（GdPDC）的PDC的动力学特征和晶体结构。酶动力学分析表明，该酶对丙酮酸具有高亲和力（pH 5时K M为0.06 mM）、高催化效率（pH 5时为1.3 • 10(6) M(-1) • s(-1)）、最适pH为5.5，最适温度为45°C。该酶不耐热（60°C时半衰期为18分钟），与其他PDC相比，单体和二聚体之间计算出的键数并未明确显示出其热稳定性相对较低的原因。其结构与运动发酵单胞菌（ZmPDC）和巴斯德醋酸杆菌PDC（ApPDC）所描述的结构高度相似，将GdPDC与ApPDC的Cα进行比较时，均方根偏差值为0.57 Å。吲哚 - 3 - 丙酮酸不是该酶的底物。在两个位点存在结构差异，涉及Thr341至Thr352区域和Asn499至Asp503区域。
这是关于重氮营养醋杆菌（PAL5）的PDC的首次研究，为未来研究其在这种内共生体中的作用奠定了基础。GdPDC的晶体结构表明该酶在进化上与运动发酵单胞菌和巴斯德醋酸杆菌的同源物密切相关，并表明存在强大的选择压力以使酶的特性保持在狭窄范围内。最适pH以及较低的热稳定性可能反映了宿主生物体的生态位以及这些特性被自然选择的条件。对吲哚 - 3 - 丙酮酸缺乏活性排除了这种脱羧酶是重氮营养醋杆菌中负责吲哚乙酸产生的酶。
