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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.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to −25°C. P355′s stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (kcat) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

Enzyme-based conversion of marine biomass to high-quality peptide ingredients is a promising strategy in bio-based industries. Despite their good nutritional profile and food grade quality, their utility in food products is limited by the fish smell. A well-known contributor to the smell is trimethylamine (TMA). Current strategies to mask or remove the odor are not effective or give rise to undesirable side effects. We have shown that trimethylamine monooxygenases (Tmms) can oxidize TMA into the odorless TMA N-oxide (TMAO) in hydrolysates.

Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways., Amination of bulky ketones, particularly in (R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S) selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R) amines for the production of high-value products and to screen for those converting putrescine. IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

This is the pre-peer reviewed version of the following article: Bjerga, G.E.K., Larsen, Ø., Arsın, H., Williamson, A., García-Moyano, A., Leiros, I. & Puntervoll, P. (2018). Mutational analysis of the pro-peptide of a marine intracellular subtilisin protease supports its role in inhibition. Proteins: Structure, Function, and Bioinformatics, which has been published in final form at https://doi.org/10.1002/prot.25528. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60 °C. The high‐resolution 3‐dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro‐peptide bound. The pro‐peptide is removed in other homologs, but the removal of the pro‐peptide from the i>Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N‐terminal residues. Furthermore, the pro‐peptide contains a conserved LIPY/F‐motif, which was found to be involved in inhibition of the catalytic activity.

Metallo--lactamases (MBLs) hydrolyze virtually all -lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative -lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with -lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the -lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Background In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. Results In our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene. Conclusion We provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.

The metallo-β-lactamases (MBLs) are enzymes with the ability to hydrolyse the β-lactam antibiotics. The worldwide emergence of the antibiotic resistant MBLs poses an increasing clinical threat. The VIM enzymes are a growing family of carbapenemases with a wide geographic distribution in Europe, South America and the USA. The VIM-7, the first VIM enzyme to be discovered in the USA, is the most divergent member of the VIM-enzymes with the closest similarity to VIM-1 with a 77% amino acid identity. The VIM-7 has a conserved D120 in the active site, which, in catalysis, plays an important role. The Y224 residue present in the VIM-2, which currently is the most widespread MBL, is though to have an impact on the activity. Three site-directed mutations of the VIM-7, with a previously solved structure, were made; D120A, D120N and H224Y respectively. All three mutants and the VIM-7 wt were sequenced, and the mutants VIM-7 D120A and VIM-7 H224Y with the VIM-7 wt, containing an N-terminal hexahis-tag and cleavage site, were produced, affinity purified and tested in a nitrocefin activity assay. The nitrocefin activity assay showed that the VIM-7 D120A mutant was inactive. In addition, the VIM-7 H224Y mutant was purified using gel filtration. A crystal was obtained of the VIM-7 H224Y mutant, and the crystal structure was solved. Enzyme kinetic studies were performed on the VIM-7 wt and the VM-7 H224Y, which resulted in the VIM-7 H224Y to have a significantly higher catalytic efficiency towards specific antibiotic substrates than the VIM-7 wt. The VIM-7 D120A mutant would not hydrolyse the antibiotic substrate ertapenem, and was not tested with other antibiotics. The inactive VIM-7 D120A and the active VIM-7 H224Y showed that by mutating one single amino acid, the D120 and the H224 residues, it has a significant effect on the activity of the VIM-7.