Characterization of the Role of Amino Acid Residues in the 2-Hydroxybiphenyl 3-Monooxygenase Catalysis Based on Bioinformatic Analysis of the Flavin-dependent Monooxygenases and Supercomputer Modeling of the Structure of Mobile Fragments Applying Variational Autoencoders
DOI:
https://doi.org/10.14529/jsfi250408Keywords:
flavin-dependent monooxygenases, 2-hydroxybiphenyl 3-monooxygenase from Pseudomonas azelaica, mobile loop structure prediction, full-length protein modeling, bioinformatics analysis, functional amino acid residuesAbstract
By modeling of predominant conformations of mobile loops in previously unresolved regions of 2-hydroxybiphenyl 3-monooxygenase structure (PDB ID: 5BRT) using GPU-accelerated metadynamics simulations integrated with artificial intelligence and high-performance computing the full-length protein model was built. Combined with bioinformatic analysis of the flavin-dependent monooxygenases it allowed to propose the functional role of amino acid residues in the 2-hydroxybiphenyl 3-monooxygenase catalysis. Three subfamily-specific residues Glu359, Lys339, Arg360 and the Asp332 residue, conservative throughout the entire family of flavin-dependent monooxygenases, form salt bridges Glu359-Lys339 and Arg360-Asp332, which stabilize alpha helices preserving the integrity of the Rossmann fold of the FAD-binding domain; subfamily-specific residues Trp338 and Glu359 provide the correct positioning of alpha-helices by interacting with two conservative residues Asp557 and Arg555 from the hydroxylase domain.
NAD binding pocket is formed by a number of subfamily-specific residues Trp38, Ser40, Ser42, Arg46, Ser47, Ala180, Asn205, Ser291, Trp293 located in an elongated pocket adjacent to the FAD binding site. The Asp313 residue, conservative in the entire family of flavin-dependent monooxygenases, directly interacts with FAD through hydrogen bonding with 2’-OH-ribitol, contributing to the binding and orientation of the cofactor. The Arg46, Ser47, Gly202, Ser203, Asn205, Arg242, Val253, Trp293, Met321, and Pro320, conservative for the entire family, play a crucial role forming the substrate binding site. The binding of cofactors and substrate in a quaternary complex and their orientation due to interactions with subfamily-specific positions Arg46, Ala180, His181 and Trp293 allows to perform the hydride transfer to the substrate stereospecifically. The triple stacking interaction between the FAD isoalloxazine ring, NADH nicotinamide ring and the subfamily-specific residue Trp293 leads to the formation of a highly stable charge-transfer complex and preferential Pro-S position in 2-hydroxybiphenyl 3-monooxygenase catalysis.
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