Mechanistic and structural studies of the non-heme iron oxygenase enzymes protocatechuate 3,4-dioxygenase, CmlA, and CmlI

Abstract

Oxygenase enzymes catalyze a remarkable variety of challenging biological transformations using molecular dioxygen (O2) as a cosubstrate. Oxygenases make possible the direct reaction of ground-state triplet O2 with organic singlet substrates, a transformation that is spin-forbidden by quantum mechanics. The set of chemical processes by which enzymes overcome this forbidden reaction are collectively termed ‘O2 activation.’ The result of oxygenase reactions is incorporation of one or both atoms of O from O2 into the organic substrate. Because of the critical role of oxygenases in many fundamental biological processes, an understanding of the catalytic and regulatory mechanisms that underlie O2 activation is required for the development of new medical, industrial, ecological and agricultural technologies. Non-heme iron oxygenases use mononuclear iron or diiron cofactors that are not coordinated in a porphyrin scaffold. This dissertation focuses on structural and mechanistic studies of three non-heme iron enzymes. The first, protocatechuate 3,4-dioxygenase (3,4-PCD), is an archetypal aromatic ring-cleaving oxygenase from soil bacteria that uses the oxidized form (Fe3+) of the iron cofactor to react with O2. It is a member of one of only two enzyme families that use Fe3+ to activate O2. CmlA is a diiron cluster-containing oxygenase that is involved in the non-ribosomal peptide synthetase-mediated biosynthesis of the antibiotic chloramphenicol. It catalyzes the first step in this pathway: the β-hydroxylation of an amino acid precursor of the antibiotic. The third enzyme, CmlI, also uses a diiron cluster cofactor and catalyzes the final step in chloramphenicol biosynthesis by converting the arylamine group of the chloramphenicol precursor to its arylnitro analog. Herein, we report the determination of the X-ray crystal structures of the two most critical oxygenated intermediates in the 3,4-PCD catalytic cycle and characterize the reaction of the enzyme with diagnostic substrates. This work confirms mechanistic proposals that have existed since the 1960s and lays the groundwork for future studies. In the last two chapters of the dissertation, we report the X-ray crystal structures of CmlA and CmlI and compare their structures to other diiron cluster-utilizing enzymes. We also discuss the insights gained into their catalytic and regulatory mechanisms.