BioTechnology Institute, University of Minnesota, 1479 Gortner Avenue, Saint Paul, MN, 55108, USA.
Present address: Department of Chemistry, Bethel University, 3900 Bethel Drive, Saint Paul, MN, 55112, USA.
Chembiochem. 2019 Jul 1;20(13):1701-1711. doi: 10.1002/cbic.201800821. Epub 2019 May 16.
Enzyme-catalyzed β-lactone formation from β-hydroxy acids is a crucial step in bacterial biosynthesis of β-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β-lactone synthetase activity using synthetic β-hydroxy acid substrates with alkene or alkyne moieties. β-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain β-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the β-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the β-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.
酶催化的β-内酰胺形成β-羟基酸是细菌生物合成β-内酰胺天然产物和膜烃的关键步骤。我们使用带有烯烃或炔烃部分的合成β-羟基酸底物开发了一种新型的连续β-内酰胺合酶活性测定法。β-内酰胺形成后迅速脱羧形成共轭三烯发色团，可通过紫外/可见光谱实时评估。该测定法用于确定植物病原体黄单胞菌的长链β-内酰胺合酶 OleC 的稳态动力学。定点突变用于测试保守活性位点残基在 Mg 和 ATP 结合中的作用。之前的报告表明 OleC 使底物的羟基腺嘌呤化。在这里，我们提出了几条证据，包括羟胺捕获 AMP 中间体，以证明在形成β-内酰胺最终产物之前，底物的羧基被腺嘌呤化。使用一组 9 种底物类似物通过 HPLC 和 GC-MS 研究了黄单胞菌 OleC 的底物特异性。2-己基-3-羟基辛酸的立体异构体被合成，并且 OleC 优先选择（2R，3S）非对映异构体，这与上下游途径酶的立体选择性一致。该生化知识用于指导β-内酰胺合酶的系统发育分析，以在酰基辅酶 A 合成酶、NRPS 腺苷酸化结构域和荧光素超家族中映射其功能多样性。
