School of Pharmaceutical Sciences, Taizhou University, 1139 Shifu Rd, Taizhou, 318000, China.
College of Life Sciences, Shanghai Normal University, 100 Guilin Rd, Shanghai, 200233, China.
Appl Microbiol Biotechnol. 2024 Sep 25;108(1):474. doi: 10.1007/s00253-024-13311-2.
(R)-3-Isobutylglutarate monoamide (R-IBM) is a key intermediate in the synthesis of the analgesic drug pregabalin. Recently, the imidase BpIH derived from Burkholderia phytofirmans was identified as a promising catalyst for the industrial production of R-IBM. Notably, this catalyst has the distinct advantage of achieving a 100% theoretical yield from 3-isobutyl glutarimide (IBI). In this study, homology modeling and structure alignment techniques were used to determine the substrate binding pocket of BpIH. Semi-rational design was used to analyze the amino acid residue conservation in the binding pocket region of BpIH. Interestingly, mutations of several low-conserved amino acid located 6-9 Å from the substrate significantly enhanced the catalytic activity of BpIH. Among them, the triple mutant Y37FH133NS226I (YHS-I) showed approximately a fivefold increase in enzyme activity and a significantly improved catalytic efficiency (k/Km). Under the same reaction time and conditions, YHS-I successfully converted IBI into R-IBM with a conversion rate of 88.87%, with an enantiomeric excess (ee) of the product exceeding 99.9%. In comparison, wild-type BpIH had a conversion rate of only 38.15%. Molecular dynamics and docking results indicated that YHS-I had higher rigidity around the mutation sites. The synergistic substitutions of Y37F, H133N, and S226I altered the interaction network within the mutation site, enhancing the protein's affinity for the substrate and improving catalytic efficiency. KEY POINTS: • 100% theoretical yield of R-IBM by BpIH compared with 50% by resolution • Semi-rational design of BpIH based on conservativity with homologous enzymes • Mutant with enzyme activity of sixfold and product ee value of 99.9.
(R)-3-异丁基戊二酰胺（R-IBM）是合成镇痛药普瑞巴林的关键中间体。最近，从植物固氮菌中分离出的亚胺酶 BpIH 被鉴定为工业生产 R-IBM 的有前途的催化剂。值得注意的是，这种催化剂具有从 3-异丁基戊二酰亚胺（IBI）获得 100%理论产率的明显优势。在这项研究中，使用同源建模和结构比对技术来确定 BpIH 的底物结合口袋。半理性设计用于分析 BpIH 结合口袋区域的氨基酸残基保守性。有趣的是，位于底物 6-9 Å 处的几个低保守氨基酸的突变显著提高了 BpIH 的催化活性。其中，三重突变体 Y37FH133NS226I（YHS-I）的酶活性提高了约五倍，催化效率（k/Km）也显著提高。在相同的反应时间和条件下，YHS-I 成功地将 IBI 转化为 R-IBM，转化率为 88.87%，产物的对映体过量（ee）超过 99.9%。相比之下，野生型 BpIH 的转化率仅为 38.15%。分子动力学和对接结果表明，YHS-I 在突变位点周围具有更高的刚性。Y37F、H133N 和 S226I 的协同取代改变了突变位点内的相互作用网络，增强了蛋白质对底物的亲和力，提高了催化效率。要点： • BpIH 实现 R-IBM 的 100%理论产率，而拆分法仅为 50% • 基于与同源酶的保守性对半理性设计 BpIH • 突变体的酶活提高了六倍，产物 ee 值达到 99.9%。
