In environments where glucose is limited, some pathogenic bacteria metabolise host derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, Open-reading frame, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding ExGH motif is altered to ExGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterisation of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog N-acetylglucosamine-bound crystal structures (2.33, 2.20 and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the ExGY motif reveals that the tyrosine binds to the N-acetyl group to select for the 'boat' conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterisation provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

中文翻译：

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来自病原体金黄色葡萄球菌的N-乙酰甘露糖胺激酶中非经典ROK家族功能的基础。

在葡萄糖受限的环境中，某些病原细菌代谢宿主产生的唾液酸作为营养来源。N-乙酰甘露糖胺激酶（NanK）是细菌唾液酸导入和降解途径的第二种酶，并使用ATP将磷酸盐添加到N-乙酰甘露糖胺中以引发该分子用于未来的途径反应。序列比对显示革兰氏阳性NanK酶属于阻遏物，开放阅读框，激酶（ROK）家族，但许多缺乏该功能预期的典型Zn结合基序，而与糖结合的ExGH基序被更改为ExGY 。结果，不清楚他们如何进行这一重要反应。在这里，我们研究金黄色葡萄球菌NanK（SaNanK），这是革兰氏阳性NanK的第一个特征。我们报告了SaNanK的动力学活性以及无配体的情况，N-乙酰基甘露糖胺结合和底物类似物N-乙酰基葡糖胺结合的晶体结构（分别为2.33、2.20和2.20Å分辨率）。这些证明，结合小角度X射线散射，SaNanK是一种二聚体，在与底物结合时采用闭合构象。对ExGY基序的分析表明，酪氨酸与N-乙酰基结合，从而选择了N-乙酰甘露糖胺的“船形”构象。此外，SaNanK具有一个堆叠的精氨酸对，其中的负残基对热稳定性和催化作用至关重要。这些组合的元素用于约束活性位点并定向底物，以代替Zn结合，这代表着与常规NanK结合的显着偏离。