Klebsiella pneumoniae, a bacterial pathogen infamous for antibiotic resistance, is included in the priority list of pathogens by various public health organizations due to its extraordinary ability to develop multidrug resistance. Bacterial fatty acid biosynthesis pathway-II (FAS-II) has been considered a therapeutic drug target for antibacterial drug discovery. Inhibition of FAS-II enzyme, enoyl-acyl carrier protein reductase, FabI, not only inhibits bacterial infections but also reverses antibiotic resistance. Here, we characterized Klebsiella pneumoniae FabI (KpFabI) using complementary experimental approaches including, biochemical, x-ray crystallography, and molecular dynamics simulation studies. Biophysical studies shows that KpFabI organizes as a tetramer molecular assembly in solution as well as in the crystal structure. Enzyme kinetics studies reveal a distinct catalytic property towards crotonyl CoA and reducing cofactor NADH. Michaelis-Menten constant (K_m) values of substrates show that KpFabI has higher preference towards NADH as compared to crotonyl CoA. The crystal structure of tetrameric apo KpFabI folds into a classic Rossman fold in which β-strands are sandwiched between α-helices. A highly flexible substrate binding region is located toward the interior of the tetrameric assembly. Thermal stability assay on KpFabI with its substrate shows that the flexibility is primarily stabilized by cofactor NADH. Moreover, the molecular dynamics further supports that KpFabI has highly flexible regions at the substrate binding site. Together, these findings provide evidence for highly dynamic substrate binding sites in KpFabI, therefore, this information will be vital for specific inhibitors discovery targeting Klebsiella pneumoniae.

肺炎克雷伯菌是一种因抗生素耐药性而臭名昭著的细菌病原体，由于其产生多重耐药性的非凡能力，被多个公共卫生组织列入优先病原体清单。细菌脂肪酸生物合成途径-II (FAS-II) 已被认为是抗菌药物发现的治疗药物靶点。抑制 FAS-II 酶、烯酰基载体蛋白还原酶 FabI 不仅可以抑制细菌感染，还可以逆转抗生素耐药性。在这里，我们使用补充实验方法（包括生化、X 射线晶体学和分子动力学模拟研究）对肺炎克雷伯菌FabI (KpFabI)​​ 进行了表征。生物物理学研究表明，KpFabI 在溶液和晶体结构中以四聚体分子组装形式组织。酶动力学研究揭示了对巴豆酰 CoA 和还原辅因子 NADH 的独特催化特性。底物的Michaelis-Menten 常数 (K _m ) 值表明，与巴豆酰 CoA 相比，KpFabI 对 NADH 具有更高的偏好。四聚体 apo KpFabI 的晶体结构折叠成经典的罗斯曼折叠，其中 β 链夹在 α 螺旋之间。高度柔性的底物结合区域位于四聚体组装体的内部。KpFabI 及其底物的热稳定性测定表明，灵活性主要由辅因子 NADH 稳定。此外，分子动力学进一步支持 KpFabI 在底物结合位点具有高度灵活的区域。总之，这些发现为 KpFabI 中高度动态的底物结合位点提供了证据，因此，这些信息对于发现针对肺炎克雷伯菌的特异性抑制剂至关重要。