Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei University of Technology, Wuhan 430068, China.
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
ACS Synth Biol. 2022 May 20;11(5):1874-1880. doi: 10.1021/acssynbio.2c00032. Epub 2022 May 6.
Enzyme entrances, which function as the first molecular filters, influence substrate selectivity and enzymatic activity. Because of low binding affinities, engineering enzyme entrances that recognize non-natural substrates is a major challenge for artificial biocatalyst design. Here, the entrance of flavonoid glycosyltransferase UGT78D2 was engineered to promote the recognition of the aglycone of etoposide, a chemotherapeutic agent. We found that Q258, S446, R444, and R450, the key residues surrounding the substrate entrance, specifically guide the flux of etoposide aglycone, which has a high steric hindrance, into the active site; this activity was inferred to be determined by the entrance size and hydrophobic and electrostatic interactions. Engineering the coordination of Q258 and S446 to increase the entrance size and hydrophobic interaction between UGT78D2 and etoposide aglycone increased the affinity by 10.10-fold and the conversion by 10%. The entrance-engineering strategy applied in this study can improve the design of artificial biocatalysts.
酶入口作为第一道分子筛选器，影响着底物的选择性和酶的活性。由于结合亲和力低，工程化能够识别非天然底物的酶入口是人工生物催化剂设计的主要挑战。在这里，黄酮糖苷转移酶 UGT78D2 的入口被工程化，以促进对化疗药物依托泊苷的糖苷配基的识别。我们发现，关键残基 Q258、S446、R444 和 R450 环绕在底物入口周围，特异性地引导具有高空间位阻的依托泊苷糖苷配基进入活性位点；这种活性推断是由入口大小和疏水相互作用和静电相互作用决定的。通过工程化 Q258 和 S446 的协调作用来增加 UGT78D2 和依托泊苷糖苷配基之间的入口大小和疏水相互作用，增加了 10.10 倍的亲和力和 10%的转化率。本研究中应用的入口工程化策略可以改进人工生物催化剂的设计。
