Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

Abstract

We present here our computational study of the experimentally elusive cyclisation step in the cofactor pyridoxal 5'-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Using both model systems and a combined QM/MM approach, our calculations suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen (N1) and the Schiff base nitrogen (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amine groups of the substrate and Arg297 and Glu81 impose substantial "strain" energy on the orientation of the cyclic intermediate in order to control its trajectory. In addition the "strain" energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions.3

Application of quantum mechanics/molecular mechanics methods in the study of enzymatic reaction mechanisms

“…Also, it is important to take into account that the flexibility allowed for the MM environment during reversible pathways simulated with QM/MM MD may lead to unrealistic changes in the overall model, considering the time‐frame of the chemical step under study. Nevertheless, single‐conformation QM/MM methods continue to provide excellent results in the study of enzymatic reactions …”

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

“…For ornithine 4,5‐aminomutase (OAM) and lysine 5,6‐aminomutase, which are both class III AdoCbl‐dependent enzymes , a role for large‐scale domain re‐orientation linked to substrate binding and C–Co bond homolysis has been proposed . OAM participates in the oxidative fermentation pathway converting d ‐ornithine to 2,4‐diaminopentanoate.…”

“…This intramolecular transferase reaction, involving a 1,2 shift of the amino group, is energetically and chemically challenging as it requires breakage of chemically inert C–H and C–N bonds . Large‐scale domain dynamics triggered by substrate binding to the pyridoxal 5‐phosphate (PLP) cofactor allow OAM to overcome the energetic barrier to the reaction . C–Co bond cleavage generates the 5′‐deoxyadenosyl radical (Ado•) and cob(II)alamin radical pair.…”

“…) . Later stages of the catalytic cycle of the OAM reaction have also been investigated using QM/MM methodology . C–Co bond activation in the modelled ‘closed’ conformation arises through a synergy of steric and electrostatic effects arising from closer interaction with a proposed 5‐deoxyadenosyl (Ado) binding motif .…”

“…), placing Ado close to the external aldimine. C–Co bond homolysis coupled with transient Ado• formation and radical transfer lead to a product‐like PLP‐bound radical intermediate, which undergoes rearrangement to facilitate abstraction of hydrogen from Ado in a final ‘ring opening’ step . In the absence of further turnover, the Ado• formed then recombines with cob(II)alamin to regenerate AdoCbl in the resting form of OAM.…”