NanoGlycobiology Unit, Department of NanoBiotechnology, Universität für Bodenkultur Wien, 1190 Vienna, Austria.
Department of Chemistry, Institute of Organic Chemistry, Universität für Bodenkultur Wien, 1190 Vienna, Austria.
Biomolecules. 2021 Nov 20;11(11):1732. doi: 10.3390/biom11111732.
Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from , a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium's cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a of 19.50 ± 3.50 µM and of 0.21 ± 0.01 s as well as a of 258 ± 38 µM and a of 0.15 ± 0.01 s were revealed. CsaB was inactive on synthetic NP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-Me, supporting the necessity of a complex acceptor substrate.
酮基丙酮酸转移酶属于广泛但研究甚少的一类酶，它们利用磷酸烯醇丙酮酸 (PEP) 将糖基丙酮酸化。丙酮酸化的糖在从 PEP 到受体的丙酮酸转移过程中通过与孔雀石绿和钼酸盐络合来检测释放的磷酸盐，建立了一种快速酮基丙酮酸转移酶测定法。为了优化该测定法用于模型 4,6-酮基丙酮酸::ManNAc-转移酶 CsaB，我们通过化学酶法制备了一种 β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-苯氧基十一烷基受体，该受体模拟了细菌细胞壁糖聚合物生物合成途径中的中间产物，CsaB 在该受体上自然具有活性，并与重组 CsaB 一起使用。最佳测定条件为 37°C 下反应 5 分钟，pH7.5，然后在 37°C 下显色 1 小时，在 620nm 处测量吸光度。通过 NMR 光谱确认生成的丙酮酸化产物的结构。使用建立的测定法，可以首次确定 4,6-酮基丙酮酸转移酶的动力学常数；当改变受体和 PEP 浓度时，发现 的值为 19.50 ± 3.50µM 和 的值为 0.21 ± 0.01s， 的值为 258 ± 38µM 和 的值为 0.15 ± 0.01s。CsaB 对合成的 NP-β-d-ManNAc 和 β-d-ManNAc-β-d-GlcNAc-1-Me 没有活性，这支持了复杂受体底物的必要性。
