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

Chrást, Lukáš; Tratsiak, Katsiaryna; Planas-Iglesias, Joan; Daniel, Lukáš; Prudniková, Tatyana; Brezovský, Jan; Bednář, David; Smatanová, Ivana Kutá; Chaloupková, Radka; Damborský, Jiřı́

doi:10.3390/microorganisms7110498

Cited by 19 publications

(19 citation statements)

References 86 publications

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“…The finding of a thermostable enzyme in a cold adapted microorganism looks contradictory at a first glance. However, despite their psychrophilic origin, the occurrence of thermophilic and thermostable enzymes in Antarctic microbes has been previously reported . To a lower extent, also Ngi1_7α‐HSDH (entry 7), whose origin, as previously mentioned, is likely close to psychrophilic bacteria, showed a moderate thermophilic behavior, with an optimum temperature at 40 °C (see Figure S5 in the Supporting Information).…”

Section: Resultsmentioning

confidence: 77%

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Bertuletti

Ferrandi²,

Marzorati³

et al. 2020

Adv Synth Catal

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Hydroxysteroid dehydrogenases (HSDHs) are valuable biocatalysts for the regio‐ and stereoselective modification of steroids, bile acids and other steroid derivatives. In this work, we investigated the substrate promiscuity of this highly selective class of enzymes. In order to reach this goal, a preliminary search of HSDH homologues in in‐house or public available (meta)genomes was carried out. Eight novel NAD(H)‐dependent HSDHs, showing either 7α‐, 7β‐, or 12α‐HSDH activity, and including, for the first time, enzymes from extremophilic microorganisms, were identified, recombinantly produced, and characterized. Among the novel HSDHs, four highly active (up to 92 U mg−1) NAD(H)‐dependent 7β‐HSDHs showing negligible similarity towards previously described 7β‐HSDHs, were discovered. These enzymes, along with previously characterized HSDHs, were tested as biocatalysts for the stereoselective reduction of a panel of substrates including two α‐ketoesters of pharmaceutical interest and selected ketones that partially resemble the structural features of steroids. All the reactions were coupled with a suitable cofactor regeneration system. Regarding the α‐ketoesters, nearly all of the tested HSDHs showed a good activity toward the selected substrates, yielding the reduced α‐hydroxyester with up to 99% conversions and enantiomeric excesses. On the other hand, only the 7β‐HSDHs from Collinsella aerofaciens and Clostridium absonum showed appreciable activity toward more complex ketones, i. e., (±)‐trans‐1‐decalone, but with interesting as well as different selectivity.

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Section: Resultsmentioning

confidence: 77%

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Bertuletti

Ferrandi²,

Marzorati³

et al. 2020

Adv Synth Catal

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“…A higher apparent melting temperature than for DsvA was thus far observed only for DmxA (T m,app ϭ 65.9 Ϯ 0.1°C), although surprisingly, this enzyme was isolated from the psychrophilic bacterium Marinobacter sp. ELB17 (17). While both DsvA01 and DsvA02 exhibited similar apparent melting temperatures (61.7 Ϯ 0.2°C and 60.5 Ϯ 0.5°C, respectively) as DsvA, the apparent melting temperature of DsvA03 (54.8 Ϯ 0.5°C) was lower by 5°C (Fig.…”

Section: Figmentioning

confidence: 88%

“…However, these residues are not essential for the catalysis since they do not make a hydrogen bond with the halogen atom and the halide ion (8). Despite the claimed necessity of two halide-stabilizing residues for HLDs to function (3,8,15), the ability to hydrolyze halogenated compounds was recently also described for enzymes with just a single primary halide-stabilizing residue: DsaA (16), DmxA (17), and DmrB (18).…”

mentioning

confidence: 99%

A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique ( S )-Enantiopreference, and High Thermostability

Chmelova

Šebestová

Lišková

et al. 2020

Appl Environ Microbiol

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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 mild thermophilic bacterium Saccharomonospora viridis DSM 43017 possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, it contains the phenylalanine (F165), which can not 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+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 the unconventional composition of catalytic residues represent a valuable source of novel biocatalysts. IMPORTANCE The present study describes a novel haloalkane dehalogenase DsvA originated from a mild thermophilic bacterium Saccharomonospora viridis DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and a (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. A sequence analysis uncovered that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single-point and one double-point mutant and identified the non-canonical halide-stabilizing residue. Our study underlines the importance of searching also for non-canonical catalytic residues in protein sequences.

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“…Often, a tunnel, maybe via a gate, 50 leading to the active site preselects and preorientates the substrates. 51 , 61 , 62 There might be several tunnels to the active site, for example, one for bigger molecules and another for smaller molecules. The active-site pocket accommodates the catalytically active residues, whereof one residue may bind the substrate covalently.…”

Section: High-tech Features Of a Biocatalystmentioning

confidence: 99%

Power of Biocatalysis for Organic Synthesis

2021

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Biocatalysis, using defined enzymes for organic transformations, has become a common tool in organic synthesis, which is also frequently applied in industry. The generally high activity and outstanding stereo-, regio-, and chemoselectivity observed in many biotransformations are the result of a precise control of the reaction in the active site of the biocatalyst. This control is achieved by exact positioning of the reagents relative to each other in a fine-tuned 3D environment, by specific activating interactions between reagents and the protein, and by subtle movements of the catalyst. Enzyme engineering enables one to adapt the catalyst to the desired reaction and process. A well-filled biocatalytic toolbox is ready to be used for various reactions. Providing nonnatural reagents and conditions and evolving biocatalysts enables one to play with the myriad of options for creating novel transformations and thereby opening new, short pathways to desired target molecules. Combining several biocatalysts in one pot to perform several reactions concurrently increases the efficiency of biocatalysis even further.

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

Cited by 19 publications

References 86 publications

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique ( S )-Enantiopreference, and High Thermostability

Power of Biocatalysis for Organic Synthesis

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