Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with ¹H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in ¹H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.

磷酸葡萄糖异构酶（PGI）属于一类酶，可催化6磷酸葡萄糖向糖6磷酸的可逆异构化。PGI在糖酵解和糖异生途径中至关重要，并被提议在真核生物中发挥额外的细胞外功能。TM1385，从先前未表征的蛋白质的磷酸葡萄糖异构酶功能海栖热袍，基于结构相似性建立PGI晶体结构和计算对接推测。动力学和比色分析与¹ H核磁共振（NMR）光谱相结合，实验证实TM1385是磷酸葡萄糖异构酶（TmPGI）。¹中溶剂交换的证据1 H NMR谱支持TmPGI异构化通过基于顺式-烯二醇的机理进行。为了确定哪些氨基酸残基对TmPGI催化至关重要，将假定的活性位点残基用丙氨酸突变并筛选活性。结果支持E281对顺式-烯二醇中间体的TmPGI形成最重要，并且可能需要H310或K422的存在来进行催化，这与以前从同源PGIs中获得的观察结果相似。但是，只有TmPGI E281A / Q415A和H310A / K422A双重突变取消了活性，表明存在多余的催化残基，并且Q415可能在E281突变后参与糖磷酸酯异构化。综合考虑，我们建议TmPGI E281直接参与顺式-烯二醇中间步骤，以及H310或K422均可促进糖环的打开和关闭。