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Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17.

ABSTRACT: Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.

SUBMITTER: Chrast L

PROVIDER: S-EPMC6920932 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17.

Chrast Lukas L Tratsiak Katsiaryna K Planas-Iglesias Joan J Daniel Lukas L Prudnikova Tatyana T Brezovsky Jan J Bednar David D Kuta Smatanova Ivana I Chaloupkova Radka R Damborsky Jiri J

Microorganisms 20191028 11

Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed ...[more]

PMID: 31661858

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Project description:Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB, remain catalytically active while employing a single halide-stabilizing residue. Here, we describe a novel haloalkane dehalogenase, DsvA, from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017, possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, DsvA contains the phenylalanine F165, which cannot stabilize the halogen anion released during the enzymatic reaction by a hydrogen bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same α-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F), and DsvA03 (W162F and F165W). Interestingly, DsvA exhibits a preference for the (S)- over the (R)-enantiomers of β-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing an unconventional composition of catalytic residues represent a valuable source of novel biocatalysts.IMPORTANCE The present study describes a novel haloalkane dehalogenase, DsvA, originating from a mildly thermophilic bacterium, Saccharomonospora viridis strain DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and an (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. Sequence analysis revealed that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single point mutants and one double point mutant and identified the noncanonical halide-stabilizing residue. Our study underlines the importance of searching for noncanonical catalytic residues in protein sequences.

Project description:Two non-pigmented, motile, Gram-negative marine bacteria designated R9SW1T and A3d10T were isolated from sea water samples collected from Chazhma Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, Russia and St. Kilda Beach, Port Phillip Bay, the Tasman Sea, Pacific Ocean, respectively. Both organisms were found to grow between 4 °C and 40 °C, between pH 6 to 9, and are moderately halophilic, tolerating up to 20% (w/v) NaCl. Both strains were found to be able to degrade Tween 40 and 80, but only strain R9SW1T was found to be able to degrade starch. The major fatty acids were characteristic for the genus Marinobacter including C16:0, C16:1ω7c, C18:1ω9c and C18:1ω7c. The G+C content of the DNA for strains R9SW1T and A3d10T were determined to be 57.1 mol% and 57.6 mol%, respectively. The two new strains share 97.6% of their 16S rRNA gene sequences, with 82.3% similarity in the average nucleotide identity (ANI), 19.8% similarity in the in silico genome-to-genome distance (GGD), 68.1% similarity in the average amino acid identity (AAI) of all conserved protein-coding genes, and 31 of the Karlin's genomic signature dissimilarity. A phylogenetic analysis showed that R9SW1T clusters with M. algicola DG893T sharing 99.40%, and A3d10T clusters with M. sediminum R65T sharing 99.53% of 16S rRNA gene sequence similarities. The results of the genomic and polyphasic taxonomic study, including genomic, genetic, phenotypic, chemotaxonomic and phylogenetic analyses based on the 16S rRNA, gyrB and rpoD gene sequence similarities, the analysis of the protein profiles generated using MALDI-TOF mass spectrometry, and DNA-DNA relatedness data, indicated that strains R9SW1T and A3d10(T) represent two novel species of the genus Marinobacter. The names Marinobacter salarius sp. nov., with the type strain R9SW1(T) ( = LMG 27497(T) = JCM 19399(T) = CIP 110588(T) = KMM 7502(T)) and Marinobacter similis sp. nov., with the type strain A3d10(T) ( = JCM 19398(T) = CIP 110589(T) = KMM 7501T), are proposed.

Dataset Information

Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17.

Publications

Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium <i>Marinobacter</i> sp. ELB17.

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