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Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.

Protein hydrolysates made from marine by-products are very nutritious but frequently contain trimethylamine (TMA), which has an unattractive fish-like smell. Bacterial trimethylamine monooxygenases can oxidize TMA into the odorless trimethylamine N -oxide (TMAO) and have been shown to reduce TMA levels in a salmon protein hydrolysate. To make the flavin-containing monooxygenase (FMO) Methylophaga aminisulfidivorans trimethylamine monooxygenase (mFMO) more suitable for industrial application, we engineered it using the Protein Repair One-Stop Shop (PROSS) algorithm. All seven mutant variants, containing 8 to 28 mutations, displayed increases in melting temperature of between 4.7°C and 9.0°C. The crystal structure of the most thermostable variant, mFMO_20, revealed the presence of four new stabilizing interhelical salt bridges, each involving a mutated residue. Finally, mFMO_20 significantly outperformed native mFMO in its ability to reduce TMA levels in a salmon protein hydrolysate at industrially relevant temperatures. IMPORTANCE Marine by-products are a high-quality source for peptide ingredients, but the unpleasant fishy odor caused by TMA limits their access to the food market. This problem can be mitigated by enzymatic conversion of TMA into the odorless TMAO. However, enzymes isolated from nature must be adapted to industrial requirements, such as the ability to tolerate high temperatures. This study has demonstrated that mFMO can be engineered to become more thermostable. Moreover, unlike the native enzyme, the best thermostable variant efficiently oxidized TMA in a salmon protein hydrolysate at industrial temperatures. Our results present an important next step toward the application of this novel and highly promising enzyme technology in marine biorefineries., Competing Interests: The authors declare no conflict of interest.

Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the environmental DNA (eDNA) was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH 0 , retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH 0 (optimum temperature [ T opt ], 50°C; melting temperature [ T m ], 55.7°C; optimum pH [pH opt ], 9.5) was stable in the presence of 10 to 20% (vol/vol) organic solvents and exhibited hydrolytic activity against p -nitrophenyl esters from acetate to palmitate, preferably butyrate (496 U mg -1 ), and a large battery of 69 structurally different esters (up to 30.2 U mg -1 ), including bis(2-hydroxyethyl)-terephthalate (0.16 ± 0.06 U mg -1 ). This broad substrate specificity contrasts with the fact that EH 0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a tight folding of its cap domain. We propose that this cap domain is a highly flexible structure whose opening is mediated by unique structural elements, one of which is the presence of two contiguous proline residues likely acting as possible hinges, which together allow for the entrance of the substrates. Therefore, this work provides a new role for the cap domain, which until now was thought to be an immobile element that contained hydrophobic patches involved in substrate prerecognition and in turn substrate specificity within family IV esterases. IMPORTANCE A better understanding of structure-function relationships of enzymes allows revelation of key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH 0 , a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH 0 revealed the potential of the sorghum rhizosphere microbiome as a source of enzymes with interesting properties, such as pH and solvent tolerance and remarkably broad substrate promiscuity. Its structure resembled those of homologous proteins from mesophilic Parvibaculum and Erythrobacter spp. and hyperthermophilic Pyrobaculum and Sulfolobus spp. and had a very narrow, single-entry access tunnel to the active site, with access controlled by a capping domain that includes a number of nonconserved proline residues. These structural markers, distinct from those of other substrate-promiscuous esterases, can help in tuning substrate profiles beyond tunnel and active site engineering.

Family VIII esterases present similarities to class C β-lactamases, which show nucleophilic serines located at the S-X-X-K motif instead of the G-X-S-X-G or G-D-S-(L) motif shown by other carboxylesterase families. Here, we report the crystal structure of a novel family VIII (subfamily VIII. I) esterase (EH 7 ; denaturing temperature, 52.6 ± 0.3 °C; pH optimum 7.0-9.0) to deepen its broad substrate range. Indeed, the analysis of the substrate specificity revealed its capacity to hydrolyse nitrocefin as a model chromogenic cephalosporin substrate (40.4 ± 11.4 units·g -1 ), and a large battery of 66 structurally different esters (up to 1730 min -1 ), including bis(2-hydroxyethyl)-terephthalate (241.7 ± 8.5 units·g -1 ) and the mycotoxin T-2 (1220 ± 52 units·g -1 ). It also showed acyltransferase activity through the synthesis of benzyl 3-oxobutanoate (40.4 ± 11.4 units·g -1 ) from benzyl alcohol and vinyl acetoacetate. Such a broad substrate scope is rare among family VIII esterases and lipolytic enzymes. Structural analyses of free and substrate-bound forms of this homooctamer esterase suggest that EH 7 presents a more opened and exposed S1 site having no steric hindrance for the entrance of substrates to the active site, more flexible R1, R2 and R3 regions allowing for the binding of a wide spectrum of substrates into the active site, and small residues in the conserved motif Y-X-X containing the catalytic Tyr enabling the entrance of large substrates. These unique structural elements in combination with docking experiments allowed us to gain valuable insights into the substrate specificity of this esterase and possible others belonging to family VIII., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris , and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli , whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae , may be an asset for development of new value chains in the biorefinery industry., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)