Structural and Biochemical Characterization of MppQ, an L-Enduracididine Biosynthetic Enzyme from Streptomyces hygroscopicus
Structural and Biochemical Characterization of MppQ, an L-Enduracididine Biosynthetic Enzyme from Streptomyces hygroscopicus
复制标题
DOI：
10.1021/acs.biochem.3c00428
复制
发表时间：
2023-10-27
期刊：
影响因子：
2.9
通讯作者：
Silvaggi，Nicholas R.
中科院分区：
文献类型：
--
作者：
Vuksanovic，Nemanja;Melkonian，Trevor R.;Silvaggi，Nicholas R.
MppQ是吸水链霉菌的一种功能未知的酶，参与非蛋白氨基酸L-耐尿酸的生物合成(L-完)。由于L-END是几种具有抗耐药病原体活性的多肽的组成部分，了解其生物合成途径将有助于开发合成新型抗生素的化学酶途径。本文报道了ShMppQ与吡哆醛-5‘-磷酸(PLP)和吡哆胺-5’-磷酸(PMP)的络合物的晶体结构。ShMppQ类似于折叠I型PLP依赖的转氨酶，如天冬氨酸转氨酶。ShMppQ的三级结构由N-末端延伸、大结构域和小结构域组成。活性部位位于大小结构域的交界处，包括同源二聚体的两个原始体的残基。我们还首次报道了MppQ的功能特性，我们将其与酶促产生的2-酮尿苷孵育，并观察到其向L末端的转化，确立了ShMppQ是L末端生物合成的终止酶。此外，我们还观察到MPQ对MPPP的副产物2-酮-5-鸟苷戊酸(即2-酮精氨酸)具有较高的亲和力，这表明MPQ可能通过将2-酮精氨酸转化为起始原料L精氨酸来提高L末端生物合成的效率。在终点分析中，对一组潜在的氨基供体底物进行了针对饱和浓度的2-酮精氨酸的转氨基活性测试。以L-鸟氨酸为供体底物，合成了大部分L-Arg。对L-Orn和2-酮精氨酸转氨化反应的稳态动力学分析表明，动力学常数与其他折叠I型氨基转移酶的氨基供体底物的动力学常数一致。
MppQ is an enzyme of unknown function fromStreptomyces hygroscopicus(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. Mostl-Arg was produced withl-ornithine as the donor substrate. Steady-state kinetic analysis of the transamination reaction withl-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.