Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil

Abstract

<jats:p>Cleavage of aromatic carbon–chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from <jats:italic>Pseudomonas</jats:italic> sp. CTN-3, with 15 of its N-terminal residues truncated (Chd<jats:sup>T</jats:sup>), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (&lt;15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. Chd<jats:sup>T</jats:sup> forms a “head-to-tail” homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His<jats:sup>117</jats:sup>, His<jats:sup>257</jats:sup>, Asp<jats:sup>116</jats:sup>, Asn<jats:sup>216</jats:sup>, and a water/hydroxide as ligands. A conserved His residue, His<jats:sup>114</jats:sup>, is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π–π interaction with Trp<jats:sup>227</jats:sup>. On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.</jats:p>

Evolution of β-lactamases and enzyme promiscuity

“…The closest characterized sequences to the B1/B2 enzymes are the dinitroanisole O-demethylases (dnhA and dnhB) and the chlorothalonil dehalogenase (Chd) (Figure 3a) (Baier, et al, 2014). The Chd structure exhibits remarkable similarity to the B2 enzyme cphA, including the L8 loop and a single metal binding site due to the H116S substitution (Catlin, et al, 2020). We therefore speculate that B2 enzymes first evolved from sequences close to Chd, which may have different functions, and then further diverged into the B1 class by reacquiring the second metal binding site.…”

“…Furthermore, in both B1 and B2 MBLs, position 121 is no longer metal binding and D221 is replaced with a cysteine that only coordinates a single metal. Examples of functional mono-and di-metal active sites are found across the MBL superfamily, and certain enzymes can function in both forms (Catlin et al, 2020, Kim et al, 2020, Lisa et al, 2017, Tu et al, 2017. In addition, depending on a monoor di-metal active sites state, these enzymes exhibit distinct chemical mechanisms (Lisa, et al, 2017).…”

Insights into the catalytic mechanism of the chlorothalonil dehalogenase from Pseudomonas sp. CTN-3

“…The catalytic mononuclear Zn(II) center in Chd resides in a slightly distorted trigonal bipyramid (TBP) geometry with His117, His257, Asp116, Asn216, and a water/hydroxide as ligands (Figure 1B). The X-ray crystal structure of Chd, in combination with kinetic data allowed us to propose a catalytic mechanism for Chd (Figure 2A) (Catlin et al, 2020). However, several aspects of this proposed mechanism are unknown.…”

“…Several bacterial strains harbor Chd genes that exhibit remarkable sequence identity (>95%) (Liang et al, 2011;Ren et al, 2011;Yue et al, 2015;Yang et al, 2019). Chd was recently structurally characterized (PDB: 6UXU at 1.96 Å) revealing an αββα-sandwich fold that is commonly observed in the β-lactamase superfamily (Catlin et al, 2020). Chd is a "head-totail" homodimer, formed between two α-helices from each monomer with a Zn(II) ion at the dimer interface that likely functions as a structural site.…”

Structural and functional identification of the uncharacterized metallo-β-lactamase superfamily protein TW9814 as a phosphodiesterase with unique metal coordination

“…Consistently, the activity of MBL proteins was changed by slight sequence differences (Bahr et al, 2021;Sunden et al, 2017), which indicates that novel enzymes can emerge by minimal mutations in the MBL superfamily. Xenobiotic pesticides are degraded by the MBL superfamily members methyl parathion hydrolase and TPN dehalogenase (Catlin et al, 2020;Dong et al, 2005). These xenobioticdegrading enzymes are assumed to have evolved recently, as pesticides began to be produced commercially in the 20th century (Baier & Tokuriki, 2014;Yang et al, 2019).…”