Catalytic role of histidine-114 in the hydrolytic dehalogenation of chlorothalonil by Pseudomonas sp. CTN-3.

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, ChdH114A exhibited catalytic activity with a kcat value of 1.07 s-1, ~ 5% of wild-type (WT) Chd, and a KM of 32 µM. Thus, His114 is catalytically important but not essential. The electronic and structural aspects of the WT Chd and ChdH114A 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 ChdH114A, 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.

Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil.

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 Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (ChdT), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<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. ChdT 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 His117, His257, Asp116, Asn216, and a water/hydroxide as ligands. A conserved His residue, His114, 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 Trp227 On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.

Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil.

Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States. Given TPN's widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotometric assay for the Zn(II)-dependent, chlorothalonil-hydrolyzing dehalogenase from Pseudomonas sp. CTN-3 (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters kcat, Km , and kcat/Km ; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a Kd of 0.17 µm, consistent with inductively coupled plasma MS data for the as-isolated Chd dimer. We observed that Chd was maximally active toward chlorothalonil in the pH range 7.0-9.0, and fits of these data yielded a pKES1 of 5.4 ± 0.2, a pKES2 of 9.9 ± 0.1 (k'cat = 24 ± 2 s-1), a pKE1 of 5.4 ± 0.3, and a pKE2 of 9.5 ± 0.1 (k'cat/k' m = 220 ± 10 s-1 mm-1). Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0. Fits of UV-visible stopped-flow data suggested a three-step model and provided apparent rate constants for intermediate formation (i.e. a k'2 of 35.2 ± 0.1 s-1) and product release (i.e. a k'3 of 1.1 ± 0.2 s-1), indicating that product release is the slow step in catalysis. On the basis of these results, along with those previously reported, we propose a mechanism for Chd catalysis.

Analyzing the function of the insert region found between the α and ß-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis.

The functional roles of the (His)17 region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHaseΔ238-257 and MbNHaseΔ219-272, were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α2ß2 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)17 motif provides an MbNHase enzyme that is ∼55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseact) but ∼28% more active when expressed in the presence of PtNHaseact. MbNHaseΔ219-272 exhibits ∼55% and ∼89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseact. Proteolytic cleavage of MbNHase provides an α2ß2 heterotetramer that is modestly more active compared to WT MbNHase (kcat = 163 ±â€¯4 vs 131 ±â€¯3 s-1). Combination of these data establish that neither the (His)17 nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.

A cobalt-containing eukaryotic nitrile hydratase.

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and ß-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αß)2 homodimer in solution, analogous to the α2ß2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.

First and second sphere interactions accelerate non-native N-alkylation catalysis by the thermostable, methanol-tolerant B12-dependent enzyme MtaC.

The corrinoid protein MtaC, which is natively involved in methyl transferase catalysis, catalyzes N-alkylation of aniline using ethyl diazoacetate. Our results show how the native preference of B12 scaffolds for radical versus polar chemistry translates to non-native catalysis, which could guide selection of B12-dependent proteins for biocatalysis. MtaC also has high thermal stability and organic solvent tolerance, remaining folded even in pure methanol.

Controlling Non-Native Cobalamin Reactivity and Catalysis in the Transcription Factor CarH.

Vitamin B12 derivatives catalyze a wide range of organic transformations, but B12-dependent enzymes are underutilized in biocatalysis relative to other metalloenzymes. In this study, we engineered a variant of the transcription factor CarH, called CarH*, that catalyzes styrene C-H alkylation with improved yields (2-6.5-fold) and selectivity relative to cobalamin. While the native function of CarH involves transcription regulation via adenosylcobalamin (AdoCbl) Co(III)-carbon bond cleavage and ß-hydride elimination to generate 4',5'-didehydroadenosine, CarH*-catalyzed styrene alkylation proceeds via non-native oxidative addition and olefin addition coupled with a native-like ß-hydride elimination. Mechanistic studies on this reaction echo findings from earlier studies on AdoCbl homolysis to suggest that CarH* selectivity results from its ability to impart a cage effect on radical intermediates. These findings lay the groundwork for the development of B12-dependent enzymes as catalysts for non-native transformations.