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Histidine kinase (HK) of two-component signal transduction system (TCS) is a potential drug target for treating bacterial infections, and most HKs are bifunctional. We have previously identified the HXXXT motif of HK in HisKA subfamily to perform the phosphatase activity, but the specific working mechanism of the threonine is not well understood. In this paper, we use the phosphate group analog BeF 3 − to capture the enzymatic intermediates between HK853 and RR468 from Thermotoga maritima during dephosphorylation, and demonstrate that the T264 site is essential for populating capable near attack conformers (NAC) between enzyme and substrate to facilitate catalysis. Importantly, mutations at this site can modulate the phosphatase activity of HK. Our results help to understand the TCS signal transduction mechanisms and provide a reference for drug design. • Capture the intermediate state of HK853 enzymatic reaction. • Construct HK853 mutants with different activities based on near attack conformation theory. • Clarify the cooperation between T and H in the H×××T motif. • Elucidate the important role of T264 in the formation of near attack conformation. [ABSTRACT FROM AUTHOR]