Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multipleturnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been characterized – but only from archaeal methanogens that are anaerobic, O₂-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O₂ at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O₂-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

中文翻译：

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耐氧、多周转 THI4 噻唑合酶的结构和功能

植物和真菌 THI4 噻唑合酶在有氧条件下通过使用活性位点 Cys 残基作为硫供体的单周转自杀反应产生硫胺噻唑部分。使用硫化物作为硫供体的缺乏活性位点 Cys（非 Cys THI4）的多周转（即催化）THI4 已被表征 - 但仅来自来自富含硫化物的栖息地的厌氧、O ₂敏感的超嗜热菌的古菌产甲烷菌。这些 THI4 更喜欢铁作为辅因子。一项对原核生物基因组的调查在来自贫硫化物栖息地的需氧中温生物中发现了非 Cys THI4，这表明在有氧、温和、低硫化物条件下多次周转 THI4 操作是可能的。这通过测试 23 种代表性非 Cys THI4s 以补充大肠杆菌而得到证实Δ thiG噻唑营养缺陷型在有氧条件下。16 个明显有活性，当通过提供 Cys 提高细胞内硫化物水平时更是如此，证明在温和温度下 O ₂存在下的催化功能并表明使用硫化物或硫化物代谢物作为硫供体。比较基因组证据将非 Cys THI4 与来自结合、运输或代谢钴或其他重金属的家族的蛋白质联系起来。确定了耐氧细菌嗜氨热弧菌 THI4的晶体结构，以探索耐氧性的分子基础。与来自 O ₂的 THI4s 的结构相比，该结构表明没有大的偏差-敏感的产甲烷菌，但与替代催化金属一致。连同补充数据，支持使用钴而不是铁。我们得出结论，催化 THI4s 确实可以在有氧条件下运行，并且插入的金属辅因子可能是空气耐受性的自然决定因素。