Stepwise Simulation of 3,5-Dihydro-5-methylidene-4H-imidazol-4-one (MIO) Biogenesis in Histidine Ammonia-lyase

Abstract

A 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) electrophilic moiety is post-translationally and autocatalytically generated in homotetrameric histidine ammonia-lyase (HAL) and other enzymes containing the tripeptide Ala-Ser-Gly in a suitably positioned loop. The backbone cyclization step is identical to that taking place during fluorophore formation in green fluorescent protein from the tripeptide Ser-Tyr-Gly, but dehydration, rather than dehydrogenation by molecular oxygen, is the reaction that gives rise to the mature MIO ring system. To gain additional insight into this unique process and shed light on some still unresolved issues, we have made use of extensive molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations implementing the self-consistent charge density functional tight-binding method on a fully solvated tetramer of Pseudomonas putida HAL. Our results strongly support the idea that mechanical compression of the reacting loop by neighboring protein residues in the precursor state is absolutely required to prevent formation of inhibitory main-chain hydrogen bonds and to enforce proper alignment of donor and acceptor orbitals for bond creation. The consideration of the protein environment in our computations shows that water molecules, which have been mostly neglected in previous theoretical work, play a highly relevant role in the reaction mechanism and, more importantly, that backbone cyclization resulting from the nucleophilic attack of the Gly amide lone pair on the π* orbital of the Ala carbonyl precedes side-chain dehydration of the central serine.

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

“…126 A very recent molecular dynamics simulation study provided great insight into the process of MIO formation in PpHAL, supporting the idea that mechanical compression from neighboring residues is required to prevent the formation of stabilizing hydrogen bonds and to enforce the correct alignment of donor and acceptor orbitals. 127 The crystal structure of PpHAL also showed for the first time the general structural features of arylalanine ammonia-lyases, homotetramers mainly built of α-helices of varying lengths 128 . Each subunit contains a globular N-terminal domain and an elongated C-terminal domain with five parallel α-helices, surrounded by six more (Figure 3).…”

Computational-Designed Enzyme for β-Tyrosine Production in Lignin Valorization

“…These so-called MIOdependent aminomutases recruit an unusual highly electrophilic moiety MIO for catalyzing the 2,3-amine shift in aromatic amino acids and present outstanding enantioselectivity. The members from this enzyme family are frequently applied for an asymmetric synthesis route without cofactor recycling to produce the optically pure β-amino acids [30,31]. On the other hand, the enzymatic synthesis of optically pure β-amino acids has been considered as an attractive strategy, compared to the chemical processes, which are limited by low yield or enantiopurity, and high cost of the waste treatment.…”

Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides

“…Each complex foldamer: Li-TryR was immersed in a box of 27,000 TIP3P water molecules that extended 12 Å away from any solute atom and Na + ions were added to ensure electrical neutrality. The MD simulation protocol at 300 K and 1 atm (NPT ensemble) was carried out as described before [28] using the pmemd_cuda. SPFP module in AMBER16.…”