Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of >100.000 members of the alpha/beta-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 mu M(-1)s(-1)) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 mu M(-1)s(-1)). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology

Application and Optimization of Bioluminescence Resonance Energy Transfer (BRET) for Real Time Detection of Protein-Protein Interactions in Transgenic \u3cem\u3eArabidopsis\u3c/em\u3e as well as Structure-Based Functional Studies on the Active Site of Coelenterazine-dependent Luciferase from \u3cem\u3eRenilla\u3c/em\u3e and its Improvement by Protein Engineering

Bioluminescence resonance energy transfer (BRET) is a biological phenomenon in some marine organisms such as Renilla reniformis and Aequorea victoria. In BRET, resonance energy from decarboxylation of coelenterazine, a substrate of Renilla luciferase (RLUC), is transferred to its acceptor such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), dependent on a distance of around 5 nm between the energy donor (RLUC) and its acceptor. The activation of the energy acceptor results in a spectral change in luminescence emission. The BRET system allows investigation of in vivo protein-protein interactions in real time. This was demonstrated with two heterodimeric interactions in transgenic Arabidopsis. In an attempt to optimize the activity and to address the reaction mechanism of the RLUC enzyme, a homology model of RLUC was obtained using a haloalkane dehalogenase, LinB, as a template. Furthermore, the homology model and the crystal structures of RLUC were docked with coelenterazine. The computational analyses suggested potential roles of catalytic triad residues (Asp120, Glu144, and His285) and substrate binding residues (N53, W121, and P220) in the active site. Mutagenesis, spectroscopy, and expression in E. coli were carried out to elucidate the reaction mechanism of RLUC and the possible roles of the residues. Moreover, the catalytic triad was probed using pharmacological tests. Using random mutagenesis, a new triple mutant was isolated, which showed increased kcat, increased half-life, and higher resistance to substrate inhibition. These results establish enzymatic characteristics of RLUC and, furthermore, suggest that the triple mutant may result in potentially advantageous properties for BRET assays, including imaging routines in Arabidopsis

Characterisation of chlorinated-hydrocarbon-degrading genes of bacteria.

Thesis (Ph.D)-University of KwaZulu-Natal, Westville, 2009.1,2-dichloroethane (DCA) is one of the most widely used and produced chemicals of the modern world. It is used as a metal degreaser, solvent, chemical intermediate as well as a fuel additive. This carcinogen is toxic to both terrestrial and aquatic ecosystems and accidental spills and poor handling has resulted in contamination of the environment. Thus far several bacteria in the Northern hemisphere have been identified that are capable of utilizing this compound as a sole carbon and energy source. This report focuses on the isolation and characterization of bacterial isolates from the Southern hemisphere that are capable of degrading DCA as well as the global distribution of the DCA catabolic route. Samples obtained from waste water treatment plants were batch cultured in minimal medium containing DCA and repeatedly sub-cultured every five days over a 25 day period. A halogen release assay was performed in order to determine whether individual isolates possessed dehalogenase activity. Confirmation of DCA utilization by bacterial isolates positive for dehalogenase activity was done by sub-culturing back into minimal medium containing DCA. Enzyme activities were confirmed with cell free extracts using all of the intermediates in the proposed DCA degradative pathway and compared to a known DCA degrading microorganism. Biochemical tests and 16SrDNA sequencing indicated that all the South African isolates belonged to the genus Ancylobacter and were different from each other. Based on enzyme activities, it was found that the South African isolates may possess a similar degradative route as other DCA degrading microorganisms. Primers based on genes involved in DCA degradation were synthesized and PCR analysis was performed. It was found that all isolates possessed an identical hydrolytic dehalogenase gene whereas the other genes in the pathway could not be PCR amplified. Southern hybridization using probes based on known genes indicated that some of the isolates had homologous genes. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis indicated that the five South African isolates of Ancylobacter aquaticus are distinguishable from each other. This study is the first report indicating that microbes from different geographical locations use similar metabolic routes for DCA degradation. The first gene of the pathway (dhlA) has undergone global distribution which may be due to widespread environmental contamination

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

Insights into enzymatic halogenation from computational studies

The halogenases are a group of enzymes that have only come to the fore over the last 10 years thanks to the discovery and characterization of several novel representatives. They have revealed the fascinating variety of distinct chemical mechanisms that nature utilizes to activate halogens and introduce them into organic substrates. Computational studies using a range of approaches have already elucidated many details of the mechanisms of these enzymes, often in synergistic combination with experiment. This Review summarizes the main insights gained from these studies. It also seeks to identify open questions that are amenable to computational investigations. The studies discussed herein serve to illustrate some of the limitations of the current computational approaches and the challenges encountered in computational mechanistic enzymology

In silico molecular analysis of novel L-specific dehalogenase from Rhizobium sp. RC1

Aims: This study presents the first structural model and proposed the identity of four important key amino acid residues, Asp13, Arg51, Ser131 and Asp207 for the stereospecific haloalkanoic acid dehalogenase from Rhizobium sp. RC1. Methodology and results: The enzyme was built using a homology modeling technique; the structure of crystallized LDEX YL from Pseudomonas sp. strain YL as a template. Model validation was performed using PROCHECK to generate the Ramachandran plot. The results showed 80.4% of its residues were located in the most favoured regions suggested that the model is acceptable. Molecular dynamics simulation of the model protein was performed in water for 10 nanoseconds in which Na+ was added to neutralize the negative charge and achieved energy minimization. The energy value and RMSD fluctuation of Ca backbone of the model were computed and confirmed the stability of the model protein. Conclusion, significance and impact of study: In silico or computationally based function prediction is important to complement with future empirical approaches. L-haloacid dehalogenase (DehL), previously isolated from Rhizobium sp. RC1 was known to degrade halogenated environmental pollutants. However, its structure and functions are still unknown. This structural information of DehL provides insights for future work in the rational design of stereospecific haloalkanoic acid dehalogenases