8IHR

Cryo-EM structure of ochratoxin A-detoxifying amidohydrolase ADH3 in complex with Phe

Summary for 8IHR

• Entry DOI: 10.2210/pdb8ihr/pdb
• EMDB information: 35453
• Descriptor: Amidohydrolase family protein, ZINC ION, PHENYLALANINE (3 entities in total)
• Functional Keywords: amidohydrolase, octamer, ochratoxin a degradtion, cryo-em structure, hydrolase
• Biological source: Stenotrophomonas acidaminiphila
• Total number of polymer chains: 8
• Total formula weight: 368108.99

Authors

Dai, L.H.,Niu, D.,Huang, J.-W.,Li, X.,Shen, P.P.,Li, H.,Hu, Y.M.,Yang, Y.,Chen, C.-C.,Guo, R.-T. (deposition date: 2023-02-23, release date: 2023-08-30, Last modification date: 2023-11-15)

Primary citation

Dai, L.,Niu, D.,Huang, J.W.,Li, X.,Shen, P.,Li, H.,Xie, Z.,Min, J.,Hu, Y.,Yang, Y.,Guo, R.T.,Chen, C.C.
Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase.
J Hazard Mater, 458:131836-131836, 2023

PubMed Abstract: Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila, termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-β-phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5-2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli-expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries.

PubMed: 37331057
DOI: 10.1016/j.jhazmat.2023.131836

Experimental method

ELECTRON MICROSCOPY (2.5 Å)