Faculty Collaboration Database

Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3. J Biol Inorg Chem 2024 Jun;29(4):427-439

Date

05/26/2024

Pubmed ID

38796812

DOI

10.1007/s00775-024-02053-1

Scopus ID

2-s2.0-85194460721 (requires institutional sign-in at Scopus site)   1 Citation

Abstract

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile; TPN) is an environmentally persistent fungicide that sees heavy use in the USA and is highly toxic to aquatic species and birds, as well as a probable human carcinogen. The chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, UniProtKB C9EBR5) degrades TPN to its less toxic 4-OH-TPN analog making it an exciting candidate for the development of a bioremediation process for TPN; however, little is currently known about its catalytic mechanism. Therefore, an active site residue histidine-114 (His114) which forms a hydrogen bond with the Zn(II)-bound water/hydroxide and has been suggested to be the active site acid/base, was substituted by an Ala residue. Surprisingly, Chd^H114A exhibited catalytic activity with a k_cat value of 1.07 s^-1, ~ 5% of wild-type (WT) Chd, and a K_M of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and Chd^H114A active sites were examined using UV-Vis and EPR spectroscopy on the catalytically competent Co(II)-substituted enzyme as well as all-atomistic molecular dynamics (MD) simulations. Combination of these data suggest His114 can quickly and reversibly move nearly 2 Å between one conformation that facilitates catalysis and another that enables product egress and active site recharge. In light of experimental and computational data on Chd^H114A, Asn216 appears to play a role in substrate binding and preorganization of the transition-state while Asp116 likely facilitates the deprotonation of the Zn(II)-bound water in the absence of His114. Based on these data, an updated proposed catalytic mechanism for Chd is presented.

Author List

Gerlich G, Miller C, Yang X, Diviesti K, Bennett B, Klein-Seetharaman J, Holz RC

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University

MESH terms used to index this publication - Major topics in bold

Biocatalysis
Catalytic Domain
Fungicides, Industrial
Halogenation
Histidine
Hydrolases
Hydrolysis
Nitriles
Pseudomonas