Bains Jasleen, Boulanger Martin J
Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, Canada V8W 3P6.
J Mol Biol. 2007 Nov 2;373(4):965-77. doi: 10.1016/j.jmb.2007.08.008. Epub 2007 Aug 19.
Xenobiotic aromatic compounds represent one of the most significant classes of environmental pollutants. A novel benzoate oxidation (box) pathway has been identified recently in Burkholderia xenovorans LB400 (referred to simply as LB400) that is capable of assimilating benzoate and intimately tied to the degradation of polychlorinated biphenyls (PCBs). The box pathway in LB400 is present in two paralogous copies (boxM and boxC) and encodes eight enzymes with the first committed step catalyzed by benzoate CoA ligase (BCL). As a first step towards delineating the biochemical role of the box pathway in LB400, we have carried out functional studies of the paralogous BCL enzymes (BCLM and BCLC) with 20 different putative substrates. We have established a structural rationale for the observed substrate specificities on the basis of a 1.84 A crystal structure of BCLM in complex with benzoate. These data show that, while BCLM and BCLC display similar overall substrate specificities, BCLM is significantly more active towards benzoate and 2-aminobenzoate with tighter binding (Km) and a faster reaction rate (Vmax). Despite these clear functional differences, the residues that define the substrate-binding site in BCLM are completely conserved in BCLC, suggesting that second shell residues may play a significant role in substrate recognition and catalysis. Furthermore, comparison of the active site of BCLM with the recently solved structures of 4-chlorobenzoate CoA ligase and 2, 3-dihydroxybenzoate CoA ligase offers additional insight into the molecular features that mediate substrate binding in adenylate-forming enzymes. This study provides the first biochemical characterization of a Box enzyme from LB400 and the first structural characterization of a Box enzyme from any organism, and further substantiates the concept of distinct roles for the two paralogous box pathways in LB400.
外源性芳香族化合物是最重要的一类环境污染物。最近在洋葱伯克霍尔德菌LB400（简称为LB400）中发现了一种新的苯甲酸氧化（box）途径，该途径能够同化苯甲酸，并且与多氯联苯（PCBs）的降解密切相关。LB400中的box途径存在两个旁系同源拷贝（boxM和boxC），编码八种酶，第一步由苯甲酸辅酶A连接酶（BCL）催化。作为阐明LB400中box途径生化作用的第一步，我们对旁系同源的BCL酶（BCLM和BCLC）与20种不同的假定底物进行了功能研究。基于BCLM与苯甲酸复合物的1.84 Å晶体结构，我们为观察到的底物特异性建立了结构原理。这些数据表明，虽然BCLM和BCLC显示出相似的总体底物特异性，但BCLM对苯甲酸和2-氨基苯甲酸的活性明显更高，结合更紧密（Km），反应速率更快（Vmax）。尽管存在这些明显的功能差异，但在BCLM中定义底物结合位点的残基在BCLC中完全保守，这表明第二壳层残基可能在底物识别和催化中起重要作用。此外，将BCLM的活性位点与最近解析的4-氯苯甲酸辅酶A连接酶和2,3-二羟基苯甲酸辅酶A连接酶的结构进行比较，为介导腺苷酸形成酶中底物结合的分子特征提供了更多见解。这项研究首次对来自LB400的Box酶进行了生化表征，也是首次对来自任何生物体的Box酶进行结构表征，并进一步证实了LB400中两个旁系同源box途径具有不同作用的概念。
