Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry

Abstract

1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k cat ‫؍‬ 0.005 s ؊1 ) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.1,2,3-Trichloropropane (TCP) is a xenobiotic compound generated from the manufacture of epichlorohydrin and is used as an industrial solvent, a paint and varnish remover, and a cleaning and degreasing agent (1). TCP can be degraded by haloalkane dehalogenase (DhaA) from Rhodococcus rhodochrous NCIMB 13064 with low catalytic efficiency (k cat ϭ 0.08 s Ϫ1 ; K m ϭ 2.2 mM) (4). DhaA shares a 47.7% protein sequence identity with the haloalkane dehalogenase LinB from Sphingobium japonicum UT26. LinB is an enzyme involved in a biochemical pathway for the degradation of the pesticide lindane (␥-hexachlorocyclohexane), where it catalyzes the conversion of 1,3,4,6-tetrachloro-1,4-cyclohexadiene to 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (16). A wide spectrum of haloalkanes can be converted by LinB; however, TCP was estimated to be a haloalkane that did not serve as a substrate for LinB when conventional analytical methods were used (6, 12). We decided to apply X-ray crystallography to determine the LinB-TCP complex structure and reveal the structural basis for inactivity.LinB was expressed and purified as described previously (16). The enzyme was crystallized at 278 K using hanging drop vapor diffusion in 0.1 M Tris buffer (pH 8.9), 18 to 20% (wt/ vol) polyethylene glycol (PEG) 6000, and 0.2 M calcium acetate. Crystals of approximately 0.4 by 0.3 by 0.04 mm 3 were transferred to the mother liquor constituents with 12.5 mM TCP and immersed for 5.5 h. Complete diffraction data were collected at European Synchotron Radiation Facility beam line ID14-2 at 100 K. The cryoprotectant was composed of 20% (vol/vol) sucrose and 20% (wt/vol) PEG 400. The data collected (to 1.6 Å) were processed using HKL (Miller index) programs DENZO and SCALEPACK (18). The structure of the complex of LinB with a halogenated substrate was solved using the coordinates of native LinB (Protein Data Bank entry 1CV2). All calculations were performed using a CCP4 suite of programs (7). The model was refined initially by the rigid-body approach and subsequently by restrained maximum-likelihood optimization. Density mod...

Isolation of novel bacteria within the Chloroflexi capable of reductive dechlorination of 1,2,3‐trichloropropane

“…Microbial transformation of 1,2,3‐TCP via oxidation with O 2 as an electron acceptor and via reduction with H 2 as an electron donor are both thermodynamically favourable (Dolfing and Janssen, 1994). Although aerobic cometabolic transformation of 1,2,3‐TCP has been demonstrated previously (Bosma and Janssen, 1998), enzymes with weak catalytic activity towards 1,2,3‐TCP oxidation have been identified (Banáš et al ., 2006; Monincová et al ., 2007), and a recombinant bacterial strain able to aerobically transform 1,2,3‐TCP to a final product of 2,3‐dichloro‐1‐propanol was reported (Bosma et al ., 2002), all attempts to isolate wild‐type cultures able to utilize 1,2,3‐TCP as a sole source of carbon or energy have failed (Bosma et al ., 2002; World Health Organization, 2003). Additionally, implementation of aerobic in situ bioremediation processes are hampered by limited ability to effectively transfer O 2 to the subsurface, and release of genetically modified bacteria in bioremediation remains controversial and subject to regulatory restrictions.…”

A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii

“…Dehalogenases, haloperoxidases, epoxide hydrolases, and hydroxynitrile lyase remove the aggregational risk by inserting prolines (PDB code 2had (54)) or charged amino acids (PDB codes 4fwb, 7 3a2m (56), 1a88 (57), 1a7u (57), 3koo (58), 2zjf (59), and 3c70 (60)) into the strand holding the His-loop, lowering their amylogenic propensity considerably, or by introducing a significant twist to this segment (PDB code 2bfn) (61). The sticky ␤-edge is buried by further, non-amylogenic ␤-strands in other ␣/␤-hydrolases like acetylcolinesterases, serine carboxypeptidases, and lipases.…”

Description of Transport Tunnel in Haloalkane Dehalogenase Variant LinB D147C+L177C from Sphingobium japonicum

“…Here we present the analysis of the overall fold, access tunnels, and docked proteinsubstrate complexes in the newly reported crystal structure of LinB D147C+L177C mutant (PDB ID: 6s06, this study) and molecular dynamics (MD) model LinB MD D147C+L177C. The tunnels were compared with previously reported crystal structures of LinB WT (PDB ID: 2bfn, [18]), LinB L177W (PDB ID: 4ibq, [19]) and LinB W140A+F143L+L177W+I211L (PDB ID: 5lka, [12]) mutants.…”