Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 North Cramer Street, Milwaukee, Wisconsin 53211, United States.
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, United States.
Biochemistry. 2023 Nov 7;62(21):3105-3115. doi: 10.1021/acs.biochem.3c00428. Epub 2023 Oct 27.
MppQ is an enzyme of unknown function from (ShMppQ) that operates in the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End). Since L-End is a component of several peptides showing activity against antibiotic-resistant pathogens, understanding its biosynthetic pathway could facilitate the development of chemoenzymatic routes to novel antibiotics. Herein, we report on the crystal structures of ShMppQ complexed with pyridoxal-5'-phosphate (PLP) and pyridoxamine-5'-phosphate (PMP). ShMppQ is similar to fold-type I PLP-dependent aminotransferases like aspartate aminotransferase. The tertiary structure of ShMppQ is composed of an N-terminal extension, a large domain, and a small domain. The active site is placed at the junction of the large and small domains and includes residues from both protomers of the homodimer. We also report the first functional characterization of MppQ, which we incubated with the enzymatically produced 2-ketoenduracidine and observed the conversion to L-End, establishing ShMppQ as the final enzyme in L-End biosynthesis. Additionally, we have observed that MppQ has a relatively high affinity for 2-keto-5-guanidinovaleric acid (i.e., 2-ketoarginine), a shunt product of MppP, indicating the potential role of MppQ in increasing the efficiency of L-End biosynthesis by converting 2-ketoarginine back to the starting material, l-arginine. A panel of potential amino-donor substrates was tested for the transamination activity against a saturating concentration of 2-ketoarginine in end-point assays. Most l-Arg was produced with l-ornithine as the donor substrate. Steady-state kinetic analysis of the transamination reaction with l-Orn and 2-ketoarginine shows that the kinetic constants are in line with those for the amino donor substrate of other fold-type I aminotransferases.
ShMppQ 是一种来自 （ShMppQ）的未知功能的酶，它在非蛋白氨基酸 L-端长素（L-End）的生物合成中起作用。由于 L-End 是几种对抗生素耐药病原体具有活性的肽的组成部分，因此了解其生物合成途径可以促进化学酶法合成新型抗生素的发展。在此，我们报告了 ShMppQ 与吡哆醛-5'-磷酸（PLP）和吡哆胺-5'-磷酸（PMP）复合物的晶体结构。ShMppQ 类似于折叠 I 型依赖 PLP 的转氨酶，如天冬氨酸转氨酶。ShMppQ 的三级结构由 N 端延伸、一个大亚域和一个小域组成。活性位点位于大小亚基的交界处，包含同源二聚体中两个亚基的残基。我们还报告了 MppQ 的首次功能表征，我们将其与酶促产生的 2-酮内长素孵育，并观察到转化为 L-End，从而确立 ShMppQ 为 L-End 生物合成的最后一个酶。此外，我们还观察到 MppQ 对 2-酮-5-胍基戊酸（即 2-酮精氨酸）的亲和力相对较高，这是 MppP 的分流产物，表明 MppQ 通过将 2-酮精氨酸转化回起始物质 L-精氨酸，可能在提高 L-End 生物合成效率方面发挥作用。我们测试了一组潜在的氨基供体底物对 2-酮精氨酸饱和浓度的转氨活性，在终点测定中。大多数 l-Arg 是用 l-Orn 作为供体底物产生的。用 l-Orn 和 2-酮精氨酸进行转氨反应的稳态动力学分析表明，动力学常数与其他折叠 I 型转氨酶的氨基供体底物的动力学常数一致。
