Crystallization and Crystallographic Analysis of a Bradyrhizobium Elkanii USDA94 Haloalkane Dehalogenase Variant with an Eliminated Halide-Binding Site

Abstract

Haloalkane dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants, for biocatalysis, biosensing and molecular tagging. The double mutant (Ile44Leu + Gln102His) of the haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeA∆Cl) was constructed to study the role of the second halide-binding site previously discovered in the wild-type structure. The variant is less active, less stable in the presence of chloride ions and exhibits significantly altered substrate specificity when compared with the DbeAwt. DbeA∆Cl was crystallized using the sitting-drop vapour-diffusion procedure with further optimization by the random microseeding technique. The crystal structure of the DbeA∆Cl has been determined and refined to the 1.4 Å resolution. The DbeA∆Cl crystals belong to monoclinic space group C121. The DbeA∆Cl molecular structure was characterized and compared with five known haloalkane dehalogenases selected from the Protein Data Bank.Crystals 2019, 9, 375 2 of 12 and the cap domain is essential for substrate specificity and recognition [6]. A deep cleft is situated between these two domains, allowing the solvent to access the buried active site. The active site is composed of two halide-anion stabilizing residues and the catalytic triad consisting of a nucleophile, a base and an acid [7]. HLDs can be divided into three subfamilies, HLD-I, HLD-II and HLD-III, according to the composition of the catalytic residues and the anatomy of the cap domain [4].A novel HLD DbeA from B. elkanii USDA94, a member of HLD-II subfamily [4], was structurally and biochemically characterized [7]. The structure of DbeA wild type was determined to 2.2 Å resolution and displays a typical topology of the α/β-hydrolases (EC 3.8.1.5). The unique feature of the DbeA structure is the presence of two halide-binding sites, both fully occupied by chloride anions [7]. The first halide-binding site is located in the protein active site and is involved in substrate binding and stabilization of halogen ion produced during dehalogenation reaction. DbeA active site consists of five catalytic residues: two halide stabilizing residues (Trp104 and Asn38) and three amino acids essential for the catalytic activity of the enzyme [2,4]: the nucleophile Asp103, the catalytic base His271, and the catalytic acid Glu127. The second halide-binding site in DbeA is unique and has never been observed within HLD structures deposited in the PDB [8]. The second halide-binding site, which is buried in the protein core domain and located approximately 10 Å far from the first halide-binding site, is formed by five amino-acid residues: Ile44, Gln274, Gln102, Gly37 and Thr40 [7]. Superposition of the DbeA structure with other related HLD-II members revealed the presence of two unique amino acids in the second halide-binding site: Gln102 instead of a typical His and Ile44 as a substitution of an ordinary Leu, thereby sufficiently increasing the cavity volume to accommodate the second halide ion. The vari...

Enzyme Engineering Renders Chlorinase the Activity of Fluorinase

“…Therefore, developing new enzymes with fluorination functions is increasingly needed. Meanwhile, numerous halogenases have been discovered in nature and are involved in the synthesis of various important natural drugs through different catalytic mechanisms. − Notably, among all halogenases, chlorinases (SalLs) share a higher sequence identity (∼35%) with FlA and possess a nucleophilic reaction mechanism analogous to that of fluorinase. SalL can accept Cl – to attack the C5′ of SAM and yield 5′-chloro-deoxyadenosine (5′-ClDA) (Scheme ).…”

Stabilization of Haloalkane Dehalogenase Structure by Interfacial Interaction with Ionic Liquids

“…Haloalkane dehalogenases belong to a large superfamily of α/β-hydrolases that catalyze hydrolytic cleavage of carbon-halogen bonds in diverse halogenated aliphatic hydrocarbons. HLDs convert haloalkanes to their corresponding alcohols, protons, and halides [3,4]. Structurally, HLDs are composed of two domains, the main domain with a central eight-stranded β-sheet flanked by six α-helices, and a conformationally flexible helical cap domain.…”

Biological Crystallization

“…This plasmid was recently identified as a putative pathogenicity marker associated with active Crohn's disease, a clinical form of the inflammatory bowel disease. Finally, in paper nº 15 Prudnikova and coworkers report the crystallization and crystal structure determination of the double mutant (Ile44Leu + Gln102His) of the haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 (DbeA∆Cl [15]. Haloalkane dehalogenases are a very important class of microbial enzymes for environmental detoxification of halogenated pollutants.…”