Computational-Based Mechanistic Study and Engineering of Cytochrome P450 MycG for Selective Oxidation of 16-Membered Macrolide Antibiotics

Abstract

MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide M-IV. The enzyme evolved to oxidize M-IV preferentially over M-III and M-VI, which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG. Density functional theory computations determined that epoxidation of the substrate hampers its ability to undergo C−H abstraction, primarily due to a loss of hyperconjugation in the transition state. Metadynamics and molecular dynamics simulations revealed a hydrophobic sugar-binding pocket that is responsible for substrate recognition/specificity and was not apparent in crystal structures of the enzyme/substrate complex. Computational results also led to the identification of other interactions between the enzyme and its substrates that had not previously been observed in the cocrystal structures. Site-directed mutagenesis was then employed to test the effects of mutations hypothesized to broaden the substrate scope and alter the product profile of MycG. The results of these experiments validated this complementary effort to engineer MycG variants with improved catalytic activity toward earlier stage mycinamicin substrates.

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil

“…In the biosynthetic pathways of these compound mixtures, some product-promiscuous enzymes often play critical roles in shaping their component ratios. Clarification of catalytic mechanism of these enzymes would enable optimization of product distribution by enzymatic engineering 45 , 46 . In sandalwood oil biosynthesis, SaSSy produces a large amount of exo - α -bergamotene, which leads to the ratio of Z - exo - α -bergamotol comparable to those of Z - α -santalol and Z - β -santalol (two most valuable components) in the oil produced by the engineered yeast constructed with SaSSy 22 .…”

Structural and Data Science-Driven Analysis to Assess Substrate Specificity of Diketopiperazine Reverse Prenyltransferase NotF: Cascade Biocatalytic Synthesis of (–)-Eurotiumin A

“…While previous studies have used statistical analysis to understand enzyme catalysis and guide engineering efforts, [66][67][68][69] this work showcases the integration of substrate profiling using MLR with IFD as a computationally cost-effective and simple interrogation of substrate scope. To accompany random, sitesaturation, 70 structure-guided, 24 and MD-guided 71,72,73 mutagenesis strategies that our group has previously relied upon, we aim to harness the predictive power of machine learning and statistical modeling to further expand our protein engineering toolbox, and develop NotF variants and other NP biosynthetic enzymes with expanded substrate scope (Figure 7). We specifically envision engineering NotF to accommodate more bulk surrounding the indole substrate to access further functionalized biosynthetic intermediates and probe downstream tailoring events of numerous indole alkaloid biosynthetic pathways.…”

Epoxidation and late-stage C-H functionalization by P450 TamI is mediated by variant heme-iron oxidizing species

“…In this system, the iterative P450 MycG performs a strict hydroxylation followed by epoxidation yielding a similar oxidation pattern to TamI L295A. 27 Although the hydroxyl group is located at a tertiary carbon with no hydrogen atom available (unlike the case of GfsF or TamI systems) abstraction of the epoxy-hydrogen or nucleophilic opening of the epoxide could still occur to generate products similar to 9 and 10 if a different active oxidant species occurs.…”

“…This marks a distinct difference from iterative P450 MycG that is not able to catalyze hydroxylation of a substrate that is initially epoxidized. 27 The ability of TamI mutants to invert reactivity in a catalyst-controlled fashion facilitates core structure diversification to produce new analogues with unique patterns of oxidation.…”