Oxidase or peptidase? A computational insight into a putative aflatoxin oxidase from Armillariella tabescens

Abstract

Aflatoxin oxidase (AFO), an enzyme isolated from Armillariella tabescens, has been reported to degrade aflatoxin B1 (AFB1). However, recent studies reported sequence and structure similarities with the dipeptidyl peptidase III (DPP III) family of enzymes and confirmed peptidase activity toward DPP III substrates. In light of these investigations, an extensive computational study was performed in order to improve understanding of the AFO functions. Steered MD simulations revealed long‐range domain motions described as protein opening, characteristic for DPPs III and necessary for substrate binding. Newly identified open and partially open forms of the enzyme closely resemble those of the human DPP III orthologue. Docking of a synthetic DPP III substrate Arg2‐2‐naphthylamide revealed a binding mode similar to the one found in crystal structures of human DPP III complexes with peptides with the S1 and S2 subsites’ amino acid residues conserved. On the other hand, no energetically favorable AFB1 binding mode was detected, suggesting that aflatoxins are not good substrates of AFO. High plasticity of the zinc ion coordination sphere within the active site, consistent with that of up to date studied DPPs III, was observed as well. A detailed electrostatic analysis of the active site revealed a predominance of negatively charged regions, unsuitable for the binding of the neutral AFB1. The present study is in line with the most recent experimental study on this enzyme, both suggesting that AFO is a typical member of the DPP III family.

Biological Detoxification of Mycotoxins: Current Status and Future Advances

“…Tomin et al ( 2019) performed an extensive computational study to investigate the affinity of an aflatoxin oxidase (AFO) from Armillariella tabescens toward AFB1, and elucidated the details of the enzyme structure. The results of this study strongly support that AFO is a typical member of the dipeptidyl peptidase III family [112].…”

New Zinc Ion Parameters Suitable for Classical MD Simulations of Zinc Metallopeptidases

“…Previous MD simulations utilizing only the electrostatic plus 12-6 LJ-type nonbonded parameters to describe interactions between the metal ion and its surroundings (some of them presented in Table ) showed that the choice of the Amber force field does not influence the Zn 2+ preference for higher (6 and above) coordination numbers during MD simulations. As a matter of fact, we performed a series of MD simulations for DPP III orthologues using either ff03, ff12SB, or ff14SB force field, − ,,− ,− and the results were consistent indicating that the zinc ion is beside by H450, H455, and E508, coordinated by E451 and two waters or substrate as well. ,, Such a strong preference for the mostly octahedrally coordinated zinc is probably boosted by the large positive charge of the zinc ion (+2 e). In addition, MD simulations of the h.DPP III complexes with natural peptides revealed a strong coordination of the zinc ion by peptides throughout the simulation, thus preventing a water molecule from entering the zinc ion coordination sphere.…”

“…It should be noted that the sequence identity between human DPP III and the other four DPP III orthologues is rather low, ranging from only 14.29% for Ca.DPP III to 40.66% for At.DPP III. 34,36,38,39 Further, human, yeast, Bt.DPP III, and mushroom At.DPPs III possess the conserved HEXXGH and EECXXE hexapeptide motifs, while in the Ca.DPP III (PDB code 6EOM) the first of them is replaced by the pentapeptide HEISH.…”

“…During MD simulations of DPPs III utilizing nonbonding parameters for the zinc ion (charge 2.0 e, while the VdW radius and energy well are 1.22 Å and 0.250 kcal/mol, respectively) ,− we had noticed a sudden change from tetrahedral to octahedral zinc coordination already at the optimization phase. Although in general the MD simulation results agreed well with the experimental results, i.e., provided accurate information on intrinsic protein dynamic and the relative ligand binding affinities, the strong preference for the octahedral zinc coordination established by one additional water molecule and carboxyl oxygen of the glutamate from the conserved HEXXGH motif − , makes the use of such parameters questionable.…”

Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action

“…Indeed, the replacement of the Cu 2+ with Zn 2+ leads to the appearance of peptidase activity of this oxidoreductase. Subsequent computer calculations by molecular dynamics and docking, together with the analysis of the surface charge distribution, confirmed that the primary AFO activity was peptidase [78], and the enzyme activity towards aflatoxin B 1 is a non-specific, secondary.…”