Ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift, interconverting d-ornithine and 2S, 4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Herein, we investigated the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, d-ornithine. Kinetic analyses revealed that residues that form salt bridges to the α-carboxylate (R297) or the α-amine (E81) of d-ornithine are most critical for OAM activity as conservative substitutions of these residues results in a 300-600-fold reduction in catalytic turnover and a more pronounced 1000- to 14,000-fold decrease in catalytic efficiency. In contrast, mutating residues that solely interact with the PLP cofactor led to more modest decreases (10-60-fold) in kcat and kcat/Km. All but one variant (S162A) elicited an increase in the kinetic isotope effect on kcat and kcat/Km with d,l-ornithine-3,3,4,4,5,5-d6 as the substrate, which indicates that hydrogen atom abstraction is more rate determining. Electron paramagnetic resonance spectra of the variants reveal that while the substitutions decrease the extent of CoC bond homolysis, they do not affect the structural integrity of the active site. Our experimental results, discussed in context with published computational work, suggests that the protonation state of the PLP cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein.
Keywords: Adenosylcobalamin; Aminomutase; PLP; Radical chemistry.
Copyright © 2017. Published by Elsevier B.V.