Pederick Jordan L, Kumar Akhil, Pukala Tara L, Bruning John B
Institute for Photonics and Advanced Sensing (IPAS), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia.
Department of Chemistry, School of Physical Sciences, North Terrace Campus, The University of Adelaide, Adelaide, South Australia, Australia.
Protein Sci. 2025 Apr;34(4):e70111. doi: 10.1002/pro.70111.
The bifunctional enzyme N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a promising antibiotic drug target, as it facilitates the biosynthesis of uridine 5'-diphospho-N-acetylglucosamine, an essential precursor of cell wall constituents. We identified that Staphylococcus aureus GlmU (SaGlmU), which was previously targeted for inhibitor development, possesses a dual-cysteine variation (C379/C404) within the acetyltransferase active site. Enzyme assays performed under reducing and non-reducing conditions revealed that the acetyltransferase activity of SaGlmU is redox-sensitive, displaying ~15-fold lower turnover and ~3-fold higher K value for the acetyl CoA substrate under non-reducing conditions. This sensitivity was absent in a C379A SaGlmU mutant. Analysis of SaGlmU by mass spectrometry, x-ray crystallography, and in silico modeling support that C379 and C404 act as a reversible, redox-sensitive switch by forming a disulfide under non-reducing conditions that impedes acetyl CoA recognition and turnover. Therefore, we recommend that future in vitro screening and characterization of SaGlmU inhibitors consider both reducing and non-reducing conditions.
双功能酶N-乙酰葡糖胺-1-磷酸尿苷转移酶（GlmU）是一个很有前景的抗生素药物靶点，因为它促进尿苷5'-二磷酸-N-乙酰葡糖胺的生物合成，而尿苷5'-二磷酸-N-乙酰葡糖胺是细胞壁成分的一种必需前体。我们发现，之前被作为抑制剂开发靶点的金黄色葡萄球菌GlmU（SaGlmU）在乙酰转移酶活性位点内存在双半胱氨酸变异（C379/C404）。在还原和非还原条件下进行的酶分析表明，SaGlmU的乙酰转移酶活性对氧化还原敏感，在非还原条件下，其周转数降低约15倍，对乙酰辅酶A底物的K值升高约3倍。在C379A SaGlmU突变体中不存在这种敏感性。通过质谱、X射线晶体学和计算机模拟对SaGlmU进行分析，结果支持C379和C404在非还原条件下通过形成二硫键发挥可逆的、对氧化还原敏感的开关作用，从而阻碍乙酰辅酶A的识别和周转。因此，我们建议未来对SaGlmU抑制剂进行体外筛选和表征时应同时考虑还原和非还原条件。
