Catalytic Role of Histidine-114 in the Hydrolytic Dehalogenation of Chlorothalonil by Pseudomonas sp. CTN-3

Authors

• Grayson Gerlich, Colorado School of Mines
• Callie Miller, Colorado School of Mines
• Xinhang Yang, Marquette University
• Brian Bennett, Marquette UniversityFollow
• Judith Klein-Seetharaman, Arizona State University
• Richard C. Holz, Colorado School of Mines

Document Type

Article

Publication Date

6-2024

Publisher

Springer

Source Publication

Journal of Biological Inorganic Chemistry

Source ISSN

0949-8257

Original Item ID

DOI: 10.1007/s00775-024-02053-1

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.

Comments

Journal of Biological Inorganic Chemistry, Vol. 29, No. 4 (June 2024): 427-439. DOI.

Recommended Citation

Gerlich, Grayson; Miller, Callie; Yang, Xinhang; Bennett, Brian; Klein-Seetharaman, Judith; and Holz, Richard C., "Catalytic Role of Histidine-114 in the Hydrolytic Dehalogenation of Chlorothalonil by Pseudomonas sp. CTN-3" (2024). Physics Faculty Research and Publications. 197.
https://epublications.marquette.edu/physics_fac/197