Abstract
Lysine dioxygenase (KDO) is an important enzyme in human physiology involved in bioprocesses that trigger collagen cross-linking and blood pressure control. There are several KDOs in nature; however, little is known about the factors that govern the regio- and stereoselectivity of these enzymes. To understand how KDOs can selectively hydroxylate their substrate, we did a comprehensive computational study into the mechanisms and features of 4-lysine dioxygenase. In particular, we selected a snapshot from the MD simulation on KDO5 and created large QM cluster models (A, B, and C) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C(4)-H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model C, the dipole moment is positioned along the C(4)-H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C(4)-H hydroxylation selectivity. Furthermore, an active site Tyr(233) residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome c peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr(233) phenol loses a proton to the nearby Asp(179) residue, while at the same time, an electron is transferred to the iron to create an iron(III)-oxo active species. This charged tyrosyl residue directs the dipole moment along the C(4)-H bond of the substrate and guides the selectivity to the C(4)-hydroxylation of the substrate.