Molecular Insights into the Regioselectivity of the Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Catalyzed C–H Hydroxylation of Amino Acids

Abstract

l-Isoleucine dioxygenase (IDO) directly catalyzes the C–H bond hydroxylation of several hydrophobic aliphatic amino acids. However, the ambiguous selectivity of IDO prevents its application in chiral hydroxy amino acid production. The hydroxylation of l-norleucine by IDO, which produces 4-hydroxynorleucine and 5-hydroxynorleucine with obvious regioselectivity, was used to investigate the mechanism of IDO regioselectivity. Along with computational structural analysis and high-throughput screening, the IDO structure revealed single-site variants (T244A, T244G, and T244S) with enhanced regioselectivity; for example, the 4-hydroxynorleucine purity in regioisomeric products increased from 78.9% (by wild-type IDO) to 95.1%, 96.6%, and 95.3%, respectively. Molecular dynamics simulations showed that mutating T244 into smaller amino acids fine-tuned the substrate binding pose. For asymmetric catalysis requiring precise positioning, this change expanded the most frequent distances between the substrate C4 or C5 and Fe2+, giving a maximum 4-hydroxynorleucine purity of 96.6%. We improved the understanding of the regioselectivity of Fe­(II)/2-ketoglutarate-dependent dioxygenases and provide a route for diversifying C–H hydroxylation-based active compounds.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes

“…This result demonstrates the trade-off phenomenon between enzyme activity and structural modulation. 53,54 In contrast, the variants Y323F, V375F, and YLDF/FYDN exhibited increased activity compared to the wild-type SsGDE. When the Tyr323 residue was replaced with phenylalanine, the catalytic activity for maize amylopectin, potato amylopectin, and DE4 maltodextrin increased by 31.73, 30.19, and 20.63%, respectively.…”

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

“…In contrast to other zones, there were three mutations in Zone 4 (N290E, E294S, S303A), implying that the residue interactions in this region may be very variable. Principal component analysis (PCA) was conducted to analyze the main motion mode of this region . The conformation clustering analysis of Zone 4 revealed that the movement of this region was more concentrated in mutants M1–1 and M2, whereas that in WT was more diffuse, with the distribution spread across three regions (Figure A–C).…”

“…Principal component analysis (PCA) was conducted to analyze the main motion mode of this region. 55 The conformation clustering analysis of Zone 4 revealed that the movement of this region was more concentrated in mutants M1−1 and M2, whereas that in WT was more diffuse, with the distribution spread across three regions (Figure 5A−C). By extracting and comparing the typical conformations, additional hydrogen bonding networks between the main chains and side chains of Zone 4 were found in both M1−1 and M2, which contribute to the "tightening" conformation of this region (Figure 5D−F).…”

Rational Design of the Substrate Tunnel of β-Ketothiolase Reveals a Local Cationic Domain Modulated Rule that Improves the Efficiency of Claisen Condensation

“…A loop region (residues 121−156) near the cationic loop that belongs to the loop domain of Tfu_0875 L163H displayed relatively high root-mean-square fluctuation (RMSF) values, indicating greater flexibility than WT-Tfu_0875 (Figure 5H). The more flexible loop region could provide a broader reaction space (4.9 vs 6.5 Å) for the substrate, conducive to extender attacking primer 41 (Figure 5I). So, we counted the number of hydrogen bonds generated between acetyl-CoA and or Tfu_0875 L163H during the whole MD process.…”