[2S03e] 07. Biocatalysis Engineering for Sustainable Bioprocessing - Part 3

6:00 PM - 6:20 PM JST(9:00 AM - 9:20 AM UTC)

[2Biocat-14] Rapid and Objective Identification of Enzyme Substrate Specificity Determinants Using EZSCAN

○Teppei Niide¹, Hiroshi Shimizu¹ (1. The University of Osaka (Japan))

Keywords:

Enzyme,Software,Substrate Specipicity

This study introduces a rapid, objective method to identify amino acid residues that control enzyme substrate specificity by comparing structurally homologous enzyme sequences. We achieved it by comparing sequence information from groups of enzymes with homologous structures. To modify enzyme functions or clarify reaction mechanisms, it is essential to identify which residues are critical. However, previous methods, such as conservation analysis, faced a challenge: they struggled to distinguish between residues required to maintain protein structure and those directly involved in function. To solve this problem, we aimed to separate functional constraint residues from evolutionary convergence, to establish and verify a prediction method, to demonstrate it through experiments, and to provide a practical tool. This method provides a rational strategy for pinpointing target residues for functional modification by leveraging evolutionary and structurally important information. Applying EZSCAN to three pairs of structurally homologous enzymes, we accurately ranked known specificity-determining residues. In trypsin/chymotrypsin, residue 172, vital for the specificity switch, ranked 1st; specificity pocket residue 189 ranked 4th, with loop residues 219 and 221 ranking 3rd and 5th. In Adenylyl Cyclase/Guanylyl Cyclase, E930 and C1002 ranked 3rd and 4th for substrate recognition; I1019, contacting the substrate, ranked 2nd. For LDH/MDH, Q81, M85, and G210 ranked 1st, 2nd, and 4th. I237, newly identified by EZSCAN, ranked 3rd among previously unreported targets. Next, the study performed experimental validation regarding the functional switch from LDH to MDH. To confirm whether the predicted residues actually contribute to function, mutations were introduced into LDH from several species, including G. stearothermophilus, L. casei, and P. falciparum. The catalytic properties of each purified LDH were evaluated. In gsLDH, the top-ranked mutation (Q86R) reduced the original LDH activity by 438 times and allowed MDH activity to appear. In lcLDH, combining the first and second mutations increased MDH activity to 1191 times that of the wild type. In pfLDH from the malaria parasite, a unique insertion sequence prevented the gain of MDH activity. However, after deleting this sequence (pfLDH_trunc) and introducing mutations, MDH activity improved gradually. This showed that functional switches sometimes require both amino acid substitutions and backbone adjustments.In summary, we established a computational method that reliably identifies specificity-determining amino acid residues in homologous enzymes, validated by application to three enzyme pairs and experimental demonstration in LDH. EZSCAN's predictions support rational enzyme modification and mechanistic studies, offering a new tool for protein engineering.