Haloalkane dehalogenases in biocatalysis:kinetic resolution and beyond

Kijk eens goed naar je eigen handen. Aan elke hand zitten precies dezelfde dingen: een duim, wijsvinger, middelvinger, ringvinger en pink, verder zijn alle botjes in je hand hetzelfde. De vorm is hetzelfde en ook zijn ze gelijk in gewicht. Maar kijk nu nog eens iets beter. Wat valt je op? Je handen mogen dan wel uit precies dezelfde onderdelen bestaan, toch zijn ze verschillend, omdat ze elkaars spiegelbeelden zijn. IJDELE SCHEIKUNDE Net als met handen lijken sommige moleculen precies op elkaar, maar zijn ze elkaars spiegelbeelden en zijn ze dus niet helemaal hetzelfde. Wel hebben ze dezelfde opbouw, met dezelfde atomen, in dezelfde volgorde en op dezelfde posities. Het gewicht is ook precies gelijk. Zulke spiegelbeelden noemen we enantiomeren; er is dan een “linker” en een “rechter” variant. Enantiomeren spelen een belangrijke rol in de biologie en scheikunde en daarmee ook in ons lichaam en in bacteriën, planten en dieren. Bij bijna alle processen in ons lichaam zijn eiwitten betrokken. Eiwit is het belangrijkste onderdeel van je haar, huid en nagels en eiwitten zijn van groot belang in je afweersysteem. Ook zijn eiwitten betrokken bij het verwerken van je voedsel. In het laatste geval werken eiwitten als katalysatoren, ze versnellen de afbraak van voedingsmiddelen tot nuttige bouwstenen en energiebronnen voor ons lichaam. In zo’n geval wordt een eiwit ook wel een enzym genoemd. Er zijn heel veel verschillende soorten enzymen die elk een eigen stofje, ook wel substraat genoemd, herkennen en omzetten naar een product. Eiwitten bestaan uit lange ketens van aan elkaar gekoppelde bouwstenen, zogeheten aminozuren. Van deze aminozuren bestaan ook twee varianten, twee enantiomeren. In de natuur wordt echter slechts één van de twee varianten gebruikt, de andere komt niet voor. Om deze reden bestaat er van een eiwit slechts één spiegelbeeld. Het blijkt dat moleculen die hetzelfde lijken, maar enantiomeren en dus elkaars spiegelbeeld zijn, een verschillend effect kunnen hebben op ons lichaam. Zo kan de biologische uitwerking van de twee enantiomeren, bijvoorbeeld van een medicijn, compleet anders zijn. Een voorbeeld hiervan is de ontstekingsremmer naproxen. Het ene enantiomeer van naproxen geeft het gewenste effect, terwijl het andere maagdarmklachten tot gevolg heeft. In zo’n geval is het erg belangrijk dat slechts één van de enantiomeren wordt toegediend

Computational Study of Protein-Ligand Unbinding for Enzyme Engineering

The computational prediction of unbinding rate constants is presently an emerging topic in drug design. However, the importance of predicting kinetic rates is not restricted to pharmaceutical applications. Many biotechnologically relevant enzymes have their efficiency limited by the binding of the substrates or the release of products. While aiming at improving the ability of our model enzyme haloalkane dehalogenase DhaA to degrade the persistent anthropogenic pollutant 1,2,3-trichloropropane (TCP), the DhaA31 mutant was discovered. This variant had a 32-fold improvement of the catalytic rate toward TCP, but the catalysis became rate-limited by the release of the 2,3-dichloropropan-1-ol (DCP) product from its buried active site. Here we present a computational study to estimate the unbinding rates of the products from DhaA and DhaA31. The metadynamics and adaptive sampling methods were used to predict the relative order of kinetic rates in the different systems, while the absolute values depended significantly on the conditions used (method, force field, and water model). Free energy calculations provided the energetic landscape of the unbinding process. A detailed analysis of the structural and energetic bottlenecks allowed the identification of the residues playing a key role during the release of DCP from DhaA31 via the main access tunnel. Some of these hot-spots could also be identified by the fast CaverDock tool for predicting the transport of ligands through tunnels. Targeting those hot-spots by mutagenesis should improve the unbinding rates of the DCP product and the overall catalytic efficiency with TCP

A single mutation in a tunnel to the active site changes the mechanism and kinetics of product release in haloalkane dehalogenase LinB

Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and, therefore, also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB, and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with the CAVER 3.0 program reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using the Random Acceleration Molecular Dynamics technique. The differences in the free energy barriers for egress of a bromide ion calculated using the Adaptive Biasing Force method are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels

ハロアルカンデハロゲナーゼの難分解性環境汚染物質分解能の機能進化に関する研究

Tohoku University永田裕二課

Redutive Dehalogenation of Chlorinated Alkanes by Novel Bacteria at the PetroProcessor of Louisiana Inc. Superfund Site

A reductively dehalogenating enrichment culture was established using chloroalkane-contaminated groundwater from the PetroProcessors of Louisiana, Inc. (PPI) Superfund site. Two novel, strictly anaerobic bacterial strains, designated as BL-DC-8 and BL-DC-9, were isolated from the enrichment culture. These strains represent the first bacteria known to anaerobically dehalogenate 1,2,3-trichloropropane, the degradation pathway of which was determined. Both strains could be cultured in completely defined basal medium and were also able to dehalogenate a variety of other vicinally chlorinated alkanes including 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, and 1,2-dichloropropane via dichloroelimination reactions. Chlorinated alkanes containing only a single chlorine substituent (1-chloropropane, 2-chloropropane), chlorinated alkenes (tetrachloroethene, trichloroethene, 1,2-dichloroethenes, vinyl chloride), and chlorinated benzenes (1-chlorobenzene and 1,2-dichlorobenzene) were not dehalogenated. Phylogenetic analysis based on 16S rRNA gene sequences showed these isolates to represent a new lineage within the Chloroflexi. Their closest previously cultured relatives are “Dehalococcoides” strains with 16S rRNA gene sequence similarities of only 90%. A quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes indicated that both strains couple reductive dechlorination to cell growth. Growth was not observed in the absence of hydrogen (H2) as an electron donor and a polychlorinated alkane as an electron acceptor. Oligonucleotide primers targeting signature 16S rRNA gene sequences of the novel isolates were used in conjunction with primers targeting “Dehalococcoides” strains to investigate spatial distribution and relative abundance of dehalogenating bacteria within the dense non-aqueous phase liquid (DNAPL) source zone at the PPI site. Results revealed the presence of bacteria similar or identical to strains BL-DC-8 and BL-DC-9 as well as “Dehalococcoides” strains throughout the DNAPL source zone. 16S rRNA gene copy concentrations as high as 1.88 ± 0.07×106 copies/mL groundwater for the isolates and 5.84 ± 0.20×105 copies/mL for “Dehalococcoides” strains. The new genus represented up to 18.6% of total bacterial 16S rRNA gene copies at some locations, and it may play an important role in natural attenuation at this site. These results have the potential to improve decision making regarding remediation strategies and may aid in development of waste treatment and monitoring approaches

Metabolism of halogenated compounds by Rhodococcus UKMP-5M

Members of the genus Rhodococcus are well known for their high metabolic capabilities to degrade wide range of organic compounds ranging from simple hydrocarbons to more recalcitrant compounds such as polychlorinated biphenyls. Their ability to display novel enzymatic capabilities for the transformation of many hazardous contaminants in the environment makes them a potential candidate for bioremediation. Rhodococcus UKMP-5M, an actinomycete isolated in Peninsular Malaysia shows great potential towards degradation of cyanide, hydrocarbons and phenolic compounds. In the present study, the capacity of this strain to degrade halogenated compounds was explored. Preliminary investigations have proven that R. UKMP-5M was not able to utilise any of the halogenated compounds tested as sole carbon and energy source, but the resting cells of R. UKMP-5M was able to dechlorinate several compounds which include chloroalkanes, chloroalcohols and chloroacids and the activity was three fold higher when the cells were grown in the presence of 1-Chlorobutane (1-CB). Therefore, 1-CB was chosen as a substrate to unravel the mechanism of dehalogenation in R. UKMP-5M. In contrast to the classic hydrolytic route for the assimilation of 1-CB in many organisms, R. UKMP-5M was able to metabolise and release chloride from 1-CB, but is unable to use the product from 1-CB metabolism as growth substrate. On comparing the protein profiles of the induced and non-induced cells of R. UKMP-5M, two types of monooxygenases were identified in the induced condition, which were not present in the uninduced sample. The strict oxygen requirement for dechlorination of 1-CB and the identification of monooxygenases in the induced protein extract suggests that 1-CB dehalogenation is likely to be catalysed by a monooxygenase. In addition to these monooxygenases, a protein that was later identified as amidohydrolase (Ah) was also found to be induced when the cells were exposed to 1-CB. Therefore, Ah from R. UKMP-5M was cloned and expressed in E. coli to test the ability of the purified Ah to release chloride from 1-CB. The heterologous expression of Ah in E. coli resulted in the formation of inclusion bodies and the western blot analyses further confirmed that no soluble form of Ah was present. Multiple attempts to obtain a soluble and functionally active Ah were not successful. Therefore, on-column refolding was carried out to obtain a biologically active Ah. A 3D model based on structural homology was predicted as a preliminary step to characterize this protein. However, when assayed with 1-CB, Ah was found not to catalyze dehalogenation. All results of this thesis suggest that metabolism of 1-CB by R. UKMP-5M is via γ-butyrolactone which acts as a potent intracellular electrophile that covalently modifies proteins and nucleic acids. The findings from this research are important to determine the metabolic capacity of a Malaysian Rhodoccoccus in dehalogenation of halogenated compounds and its potential application in bioremediation.Open Acces