Increased Thermostability of an Engineered Flavin-Containing Monooxygenase to Remediate Trimethylamine in Fish Protein Hydrolysates

Increased Thermostability of an Engineered Flavin-Containing Monooxygenase to Remediate Trimethylamine in Fish Protein Hydrolysates Marianne Goris,aIsabel Cea-Rama,bPål Puntervoll,aRasmus Ree,aDavid Almendral,cJulia Sanz-Aparicio,bManuel Ferrer,c Gro Elin Kjæreng Bjergaa a NORCE Climate & Environment - NORCE Norwegian Research Centre, Bergen, Norway b Instituto de Quimica Fisica Rocasolano (IQFR), CSIC, Madrid, Spain c Instituto de Catalisis y Petroleoquimica (ICP), CSIC, Madrid, Spain ABSTRACT 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. KEYWORDS flavin-containing monooxygenases, trimethylamine, protein hydrolysate, enzyme engineering, PROSS Flavin-containing monooxygenases (FMOs, EC 1.14.13.8) are enzymes that insert one molecule of oxygen into organic substrates using the cofactors flavin adenine dinucleotide (FAD) and NAD(P)H (1–3). A subgroup of bacterial FMOs oxidize trimethylamine (TMA) to trimethylamine N-oxide (TMAO) (Fig. 1) and are often referred to as trimethylamine monooxygenases (Tmms) (4–6). These and other FMOs have also gained interest for their ability to convert indole into the dye indigo and the drug agent indirubin (5, 7–9). TMA is a well-known contributor to the odor of spoiled fish (10) and may accumulate to give rise to a strong bodily odor in humans with trimethylaminuria (fish odor syndrome) caused by impairments in the FMO3 gene (11). Fish protein hydrolysates made from by-products from fisheries and aquaculture are of high nutritional value and have a great potential for the human consumption market Editor Marina Lotti, University of MilanoBicocca Copyright © 2023 Goris et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Gro Elin Kjæreng Bjerga, [email protected]. The authors declare no conflict of interest. Received 10 March 2023 Accepted 8 May 2023 Published 24 May 2023 June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 1 ENZYMOLOGY AND PROTEIN ENGINEERING Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. (12, 13). However, fish protein hydrolysates frequently suffer from an off-putting malodor that is mainly caused by TMA. Currently, the TMA malodor may be handled by odor masking, vaporization, encapsulation, or filtration, albeit with various degrees of success and possibly also compromising other qualities in the products. The application of Tmm enzymes is thus an alternative and novel strategy to convert TMA to the odorless TMAO in fish protein hydrolysates. This has the potential to significantly improve the organoleptic quality of fish protein hydrolysates and thereby promote their application as food ingredients, while simultaneously maintaining their nutritional profile. In a previous study, we screened 45 bacterial Tmms for their ability to oxidize TMA to TMAO (6) and identified the Methylophaga aminisulfidivorans Tmm (mFMO) (5) as a suitable candidate for application on a TMA-containing salmon protein hydrolysate. In industrial fish protein hydrolysis, enzymes are required to perform at pHs of around 6 and temperatures ranging from 45°C to 60°C (14, 15). This implies that mFMO, with an optimal temperature of 44.0°C and melting temperature of 46.7°C, would benefit from enzyme engineering to increase its stability (6). In that respect, a previous effort to engineer mFMO is encouraging. Lon car and colleagues used the computational protocol FRESCO (16, 17) to predict two mutations in mFMO, M15L (a mutation of M to L at position 15) and S23A, that when combined increased the apparent melting temperature by 3.0°C (16). The mFMO enzyme forms a dimer, and each monomer consists of two domains: the larger FAD-binding domain and the smaller NADPH-binding domain (18, 19). Upon binding, NADPH reduces the tightly bound FAD, thus generating the reactive flavin intermediate C4a-hydroperoxy-FAD and NADP 1 . The latter stabilizes the activated flavin intermediate, and together with the tyrosine residue at position 207, it shields the active site and the intermediate from the solvent (18). When entering the active site, the substrate displaces NADP 1 and is subsequently oxidized by the activated flavin intermediate. Protein Repair One-Stop Shop (PROSS) is a Web server that takes a protein structure as input and outputs several mutated sequences that are expected to have increased stability (20). PROSS combines multiple independently stabilizing mutations by integrating Rosetta modeling and phylogenetic sequence information (20). In a recent community-wide experimental evaluation of PROSS, designs for 9 of 10 tested protein targets displayed increases in temperature stability that ranged from 8.3°C to 27.0°C (21). In the present study, we employed the PROSS algorithm on mFMO to improve its thermal stability. Seven combinatorial mutant variants of mFMO, containing 8 to 28 mutations, were analyzed for their temperature stability and compared to wild-type mFMO. We demonstrate that all mFMO variants were more thermostable than the wild type. The most thermostable variant was analyzed by steady state kinetics and compared to the wild type without identifying substantial modification of the kinetic parameters. Moreover, this stabilized mFMO variant also converted TMA to TMAO more efficiently than native mFMO in a salmon protein hydrolysate at two industrially relevant temperatures, 50.0°C and 65.0°C. Finally, the crystal structure of the most thermostable variant was solved to elucidate the structural basis for the increased thermal stability, revealing loss of flexibility through a novel network of polar interactions as the main contributing factor. RESULTS Design and expression of mutant variants of mFMO with predicted increased stability. To make a more stable and temperature-resistant mFMO, ideally withstanding at least 50°C in industrial applications, we employed computational enzyme engineering. FIG 1 Chemical reaction catalyzed by trimethylamine monooxygenases. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 2 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. The most recent crystal structure of mFMO in complex with the cofactors FAD and NADP 1 (PDB identification code [ID] 2XVH)(19) was used as input to the PROSS Web server (20), along with instructions to exclude residues in contact with the cofactors, as well as dimer interfaceresidues,asmutationaltargets.PROSSproposed7mFMOvariantswiththenumber of mutations ranging from 8 to 28 (Fig. S1 in the supplemental material). The variants were named mFMO_n,wherenindicates the number of mutations. The mutations were located at or near the surface, and the number of residues predicted to form new stabilizing salt bridges increased from 2 in mFMO_8 to 8 in mFMO_28 (Table 1). In the models of mFMO_8 through mFMO_20, all new salt bridges were predicted to form between one mutated and one native residue (Table 1). The last two variants displayed more complex salt bridge patterns, including direct interactions between mutated residues: in mFMO_24, the newly introduced N394K mutation is predicted to form salt bridges with both T370D and D374, and in mFMO_28, the newly introduced P391D mutant is predicted to form a third salt bridge with N394K (Table 1). Interestingly, the majority of the new salt bridges are predicted to form interhelical connections (Fig. 2, Table 2). The number of predicted salt bridges that connect secondary structure elements gradually increases from 1 in mFMO_8 to 6 in mFMO_24 and mFMO_28. All 7 mFMO mutant variants were expressed with a C-terminal hexahistidine tag, at levels comparable to that of native mFMO, purified (Fig. S2), and verified by mass spectrometry. When expressing mFMO in Escherichia coli, the culture medium turns blue due to the enzymatic conversion of endogenous indole to indigo (5–7). The fact that the culture media of all mFMO variants turned blue following overnight expression suggested that the expressed mFMO variants were functional. Moreover, all purified mFMO variant enzymes were colored bright yellow, indicating the presence of bound FAD cofactor, which is required for function. mFMO mutant variants are functional and more thermostable than native mFMO. To investigate whether the mFMO variants had increased thermal stability compared to that of the native enzyme, we conducted protein melting studies using circular dichroism (CD). The melting temperature (T m ) of native mFMO was measured to be 46.2°C (Table 2), which is in line with the previously reported T m of 46.7°C (6). All mFMO variants demonstrated increased temperature stability compared to that of the wild-type enzyme, as reflected by their T m values, which ranged from 50.9°C for mFMO_28 to 55.2°C for mFMO_20 (Table 2). The melting temperature increased with the number of mutations from mFMO_8 to mFMO_20 but declined slightly for mFMO_24 and mFMO_28. To study the increased temperature stability of the mFMO variants further, we evaluated their residual catalytic activity against TMA after 1-h incubations at temperatures from 30.0°C to 54.0°C. The temperature at which half the enzyme activity was lost (T 50 ) ranged from 45.1°C for mFMO_8 to 50.0°C for mFMO_20, all outperforming native mFMO, TABLE 1 Predicted new salt bridges formed between mutated residues and native or mutated residues Mutated residue Residue forming a predicted salt bridge in indicated mutant mFMO variant a mFMO_8 mFMO_11 mFMO_14 mFMO_15 mFMO_20 mFMO_24 mFMO_28 N290D R292 R292 R292 R292 R292 M353K E357 E357 K358R D374 D374 D374 D374 D374 L360E 2222 R356/R292 A365D K401 K401 K401 T370D 22222N394K N394K N378D K300 K300 K300 K300 K300 K300 K300 P391D N394K N394K T370D/D374 T370D/D374/P391D L398K E366 E366 E366 E366 E366 2 Total b 234457 8 a Empty cells indicate the absence of the mutated residue listed to the left. Residues (native or mutant) forming a predicted salt bridge with the mutant residue (first column) are listed, and if a mutant residue does not form a salt bridge in an mFMO variant, it is marked by a minus sign (2). b The total numbers of mutant residues involved in forming salt bridges are shown. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 3 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. FIG 2 Predicted interhelical salt bridges involving or induced by mutated residues in PROSS mutant models. (A) The structure of the native mFMO dimer (PDBID 2XVH), which was used to generate the PROSS mutant models, is shown to the left. Chain A is shown as a ribbon model, and chain B is shown in surface view. The large domain is shown in gray, the small domain in wheat, and the cofactors FAD and NADP 1 are shown in yellow and green sticks, respectively. The figure to the right shows the native mFMO chain A rotated 260° around the yaxis to the right to emphasize the region of the structure where interhelical salt bridges are introduced in the PROSS mutants (dotted square). The emphasized region (residues 274 to 407) includes beta strands b 17 to b 20 and alpha helices a 5to a 8. (B) The predicted new interhelical salt bridges of the 7 PROSS mutant models are shown using the region and orientation described in the legend to panel A. The two models mFMO_14 and mFMO_15 have identical interhelical salt bridges and are represented by one structural model. Residues forming salt bridges are shown as sticks, hydrogen bonds between interacting residues are shown as tealcoloreddashes,andoxygenandnitrogenatomsarecoloredredandblue,respectively.Nativeandmutatedresiduesarecoloredblue and orange, respectively. Salt bridge residues that are not present in native mFMO or the previous variant are labeled. The N378D-K300D351 salt bridge, which is present in all models, connects helices a 6and a 7viathe b 18b 19 loop. The L360E-R292 salt bridge, present in mFMO_28, is not interhelical but connects helix a 6tothe b 17b 18 loop. All salt bridges that were introduced by PROSS are present in the consecutive models, except for L398K-E366, introduced in mFMO_11, which is not present in mFMO_28. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 4 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. which had a T 50 of 40.6°C (Table 2). The T 50 values increased with the number of mutations in the same manner as the T m , with a moderate decline recorded for mFMO_24 and mFMO_28. As production of protein hydrolysates is often performed between pH 6.0 and 7.0 (14, 15), we also assessed whether engineering altered the pH optimum, which was previously determinedtobe8.5fornativemFMO(6).AsseenbytheresultsinTable2,allmFMOvariantshad pH optima between 7.5 and 8.0, slightly lower than that of native mFMO. Although mFMO_20 did not have the lowest pH optimum among the mutant variants, it displayed the greatest improvement in temperature stability, as reflected by both T m and T 50 . As mFMO_20 thus emerged as the most promising variant for industrial application, we determined its optimal temperature (T opt ) for enzymatic activity and compared it to that of native mFMO. To determine the T opt , we assessed the specific activity against TMA at temperatures between 22°C and 50°C and pH 8.0 (Fig. S3). The T opt for both native mFMO and mFMO_20 was found to be 40°C (Table 2). The T opt for native mFMO was previously reported to be 45°C (at pH 7.5) (6), but the observed differences in the activities measured at 40 and 45°C in both studies were marginal. Engineering enzymes to increase stability often comes with a trade-off of diminished catalytic activity (22). We therefore performed a steady-state kinetic analysis of native mFMO and mFMO_20, using TMA as the substrate with a fixed concentration of NADPH (Table 2). Under the conditions tested, the KTMA mof native mFMO was 1.07 m M and the kTMA cat was 1.28 s 21 . The KTMA mvalue of mFMO_20 was 0.83 m M and the kTMA cat value was 0.93 s 21 , both slightly lower than those of native mFMO. Interestingly, the catalytic efficiency (k cat /K m ) of mFMO_20 remained almost identical to that of native mFMO (Table 2). mFMO_20 reduces the TMA level in salmon protein hydrolysate by 95% at 65°C. Since mFMO_20 demonstrated the most prominent increase in thermal stability, we compared its ability to convert TMA to TMAO in a salmon protein hydrolysate to that of native mFMO. The cofactor NADPH was supplemented, as the hydrolysate did not contain sufficient amounts to drive the enzymatic reaction (6). Heat-calibrated enzymes and 0.5 mM NADPH were added to salmon protein hydrolysates (pH 6.1) and incubated for 1 h at 30°C, 50°C, and 65°C, followed by measurements of TMA and TMAO concentrations (Fig. 3). When treated with the native enzyme, the TMA level in the hydrolysate was reduced by 52% at 30°C and 46% at 50°C, and only 29% reduction was observed at 65°C. The mutant variant mFMO_20 outperformed native mFMO at all temperatures, with a striking 95% reduction of TMA at both 50°C and 65°C. Network of novel polar interactions stabilizes mFMO_20. To understand the structural basis for the increased thermostability of mFMO_20, we crystallized it with the cofactors FAD and NADPH. The crystals were indexed in the C222 1 spacegroupandcontained the biological dimer within the asymmetric unit, with one FAD and one NADP 1 molecule TABLE 2 Biochemical parameters of mFMO and mutant variants Enzyme Mean value ±SD or as indicated T m ±95% CI (°C) a T 50 (°C) b T opt (°C) c Optimal pH d K m (mM) e k cat (s 21 ) e k cat /K m (mM 21 s 21 ) e mFMO 46.2 60.2 40.6 60.4 40 8.5 1.07 60.13 1.28 60.03 1.20 60.12 mFMO_8 51.2 60.1 45.1 60.9 7.5 mFMO_11 51.7 60.1 47.1 61.2 7.5 mFMO_14 53.9 60.2 49.0 61.5 8.0 mFMO_15 54.5 60.1 49.6 60.4 7.5 mFMO_20 55.2 60.1 50.0 60.4 40 8.0 0.83 60.02 0.93 60.05 1.11 60.03 mFMO_24 52.0 60.1 49.2 60.2 7.5 mFMO_28 50.9 60.1 48.9 60.5 8.0 a Melting curves were obtained by CD at pH 7.5, and melting temperature was estimated by four-parameter logistic regression of the melting curve. b The temperature where half of the enzyme activity was lost (T 50 ) was measured at pH 7.5, and the reported data are the mean values 6standard deviations (SD) from three independent experiments. c Optimal temperature (T opt ) was determined at pH 8.0 for mFMO and mFMO_20. d Optimal pH for TMA conversion was determined at 22°C. e Steady-state kinetic measurements were performed with various concentrations of TMA (Sigma-Aldrich) as the substrate and a fixed NADPH concentration (200 m M) at 23°C, pH 8.0. Reported data are the mean values 6SD from two biological replicates. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 5 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. bound per catalytic site. The crystal structure of the mFMO_20/FAD/NADP 1 complex was solved at 1.62-Å resolution, revealing a structure highly similar to that of native mFMO (PDB ID 2XVH)(19), as reflected by a calculated root mean square deviation (RMSD) of 0.27 Å (on 445 C a atoms). The small domain contains 4 mutations, and the large domain contains the other 16 (Fig. 4). Compared to native mFMO, mFMO_20 has a net charge change of 24. Interestingly, half of the mFMO_20 mutations are located in a 46-amino-acid subsequence FIG 3 Enzymatic TMA conversion in salmon protein hydrolysate. Salmon protein hydrolysates (pH 6.1) were incubated for 1 h at 30, 50, and 65°C with no enzyme (control), mFMO, or mFMO_20, all supplemented with 0.5 mM NADPH. TMA and TMAO levels were determined using UHPLC with the EVOQ Elite triple quadrupole mass spectrometer. The experiment was performed with three biological replicates, and the plot shows the mean TMA (gray) and TMAO (yellow) levels (ppm) in stacks with standard deviations (SD) represented by error bars. FIG 4 Crystal structure of mFMO_20. The structure of the mFMO_20 monomer (PDB ID 8B2D) (chain A) is shown as a ribbon model, with the large domain colored in gray and the small domain colored in wheat. The cofactors FAD and NADP 1 are shown as yellow and green sticks, respectively. The side chains of the 20 mutated residues are represented as orange sticks, and oxygen and nitrogen atoms are colored red and blue, respectively. The alpha carbons of the introduced glycine residues are shown as spheres. The 5 residues that are new compared to the sequence of mFMO_15 are labeled in green. The three a -helices where the 10 of 20 mutated residues are located are labeled in red. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 6 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. (M353Q to L398K) of a region in the large domain containing three helices: a 6 (K345 to T361), a 7 (A365D to M382), and a 8 (I390 to N406) (Fig. 3). Structural analysis revealed that five of these mutated residues form new salt bridges involving six native residues (K358R-D374, L360E-R356, A365D-K401, N378D-K300-D351, and L398K-E366), four of which form interhelical interactions(Fig.5A,Table3),thusconfirming the PROSS model predictions (Fig. 2). Helices a 6 and a 7 are directly connected by K358R-D374 and indirectly connected, via the b 18b 19 loop, by N378D-K300-D351. Helices a 7and a 8 are connected by A365D-K401 and L398KE366. Two new hydrogen bonds involving side chains are also introduced, one forming an intrahelical bond ( a 6; M353Q-R356) and the other an interhelical bond ( a 7a 8; T370D-N394) (Table 3). In addition to the salt bridges directly introduced by mutated residues, mFMO_20 has 4 salt bridges involving native-only residues that are not present in the native mFMO structure (PDB ID 2XVH) that we used as starting point (Fig. 5A). However, three of these salt FIG 5 Stabilizing salt bridges in native mFMO and mutant mFMO_20. (A) Chain A of the mFMO_20 structure is shown as a ribbon model, with the small and large domains colored in wheat and gray, respectively, and the cofactors FAD and NADP 1 shown as yellow and green sticks, respectively. Residues forming salt bridges that are unique to mFMO_20 are shown as sticks, hydrogen bonds between interacting residues are shown as teal-colored dashes, and oxygen and nitrogen atoms are colored red and blue, respectively. Native and mutated residues are colored blue and orange, respectively. The residues of salt bridges involving mutant residues are labeled. The three alpha helices where the mutated residues are located are labeled in red. (B) Chain A of the mFMO structure (PDB ID 2XVH) is visualized essentially as described in the legend to panel A. Residues that form salt bridge pairs in both the native mFMO and mutant mFMO_20 are colored slate blue, and the lone pair which is unique to mFMO is colored cyan. (C) The biological dimer of native mFMO is shown in B-factor putty representation. Red colors and large diametersofthetubesindicateflexible regions with higher B-factors, in contrast to blue colors with small diameters, indicating well-ordered regions with lower B-factors. The location of the loop between b -strands 11 and 12 is indicated. Chain B is colored white. (D) The biological dimer of mutant mFMO_20 is visualized as described in the legend to panel C. The view of all structures has been rotated 260° around the yaxis compared to the view in Fig. 4. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 7 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. bridges are present in at least 4 of the other 11 mFMO PDB structures. The presence of the apparently unique R19-E26 salt bridge in mFMO_20 may reflect differences in crystallization conditions. All but one of the salt bridges identified in native mFMO are also present in mFMO_20 (Fig. 5B). The B-factor profile of mFMO_20 is different from that of native mFMO (Fig. 5C and 5D). The lower B-factors observed near the entrance to the active site in our crystallographic analysis are consistent with the increased thermal and kinetic stability of this design. The region with the highest B-factor in mFMO_20 is the loop between b -strands 11 and 12, which is located at the entrance of the active site. In contrast, several loops in the native mFMO display higher B-factors than the b 11b 12 loop. DISCUSSION We have previously shown that mFMO can convert the malodorous TMA molecule into the odorless TMAO in a salmon protein hydrolysate (6). To make mFMO more suitable for such industrial applications, which typically take place at temperatures ranging from 45°C to 60°C (14, 15), we employed the PROSS algorithm to improve its thermal stability. All 7 mutant variants of mFMO were functional enzymes with increased thermostability (Table 2). The best variant was mFMO_20, which displayed the highest increase in temperature stability (Table 2). The crystal structure of mFMO_20 demonstrated that the overall structure of this mutant variantwashighlysimilartothatofnativemFMO,butitalsorevealednewstructuralfeatures that could explain the increased structural stability. The most striking new features were five salt bridges involving mutated residues, four of which formed stabilizing interhelical connecting bridges (Fig. 5A). Although PROSS failed to correctly model the intrahelical salt bridge between L360E and R356 and predicted an intrahelical salt bridge between N290D and R292 that was not observed in the crystal structure, all four interhelical salt bridges were correctly modeled (Fig. 2), thus emphasizing the quality of the PROSS predictions. The only interhelical salt bridge that was present in all 7 PROSS models was the N378D-K300-D351 bridge, which connects a 6to a 7viathe b 18/ b 19 loop (Fig. 2). The N378D mutation leads to the replacement of a hydrogen bond between N378 and K300 in the native mFMO structure by a stronger salt bridge between N378D and K300 and also induces K300 to form a salt bridge with D351, which is not present in the native structure. The facts that the largest increase in temperature stability from one variant to the next was observed going from the native mFMO to mFMO_8 and that mFMO_8 only contains one interhelical salt bridge may suggest that N378D is a key stabilizing mutation. However, it is difficult to deconvolute the effect of the introduced interhelical salt bridges from the effects of the other mutations. Nevertheless, the observed gradual increase in temperature stability from mFMO_8 to mFMO_20 coincides with a gradual increase in the number of such bridges from 1 in mFMO_8 to 4 in mFMO_20 (Fig. 2), suggesting that these electrostatic interactions have an important role in increasing temperature stability. These results are in line with the recently published community-wide PROSS evaluation, where a correlation between the number of mutations and gain of thermal stability was observed (21). However, despite introducing 4 and 8 more mutations, leading to TABLE 3 New polar interactions directly involving or induced by side chain atoms of mutated residues in mFMO_20 Hydrogen donor a Hydrogen acceptor a Distance (Å) b Structural location c Donor Acceptor K300 (N z ) D351 (O d 2 ) 3.0 Loop b 18b 19 a 6 K300 (N z ) N378D (O d 2 ) 2.8 Loop b 18b 19 a 7 R356 (N « /N h 2 ) M353Q (O « 1 ) 2.8/2.7 a 6 a 6 R356 (N h 1 ) L360E (O « 2 ) 3.4 a 6 a 6 K358R (N h 1 /N h 2 ) D374 (O d 2 ) 3.0/2.8 a 6 a 7 N394 (N d 2 ) T370D (O d 1 ) 3.4 a 8 a 7 L398K (N z ) E366 (O « 2 ) 3.1 a 8 a 7 K401 (N z ) A365D (O d 2 ) 2.7 a 8 a 7 a Hydrogen donor and acceptor atoms of the amino acid side chains are indicated in parentheses. b The distances between hydrogen donor and acceptor atoms of the amino acid side chains are shown. c The structural location is indicated by the secondary structure element. Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 8 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229. two more salt bridges predicted to connect secondary structure elements in mFMO_24 (both between a 7and a 8) and mFMO_28 (one between a 7and a 8 and one between a 6andthe b 17/ b 18 loop), the temperature stability of these variants decreased slightly compared to the temperature stability of mFMO_20. One possible explanation is that the intricate networks of salt bridges in mFMO_24 and mFMO_28, which also involve direct bridges between mutated residues, are incorrectly predicted by PROSS. In a previous effort to identify mutations that confer increased thermostability to mFMO, Lon car and colleagues used the FRESCO protocol to predict stabilizing single mutations (16). The FRESCO analysis yielded 140 single-mutant candidates that were expressed, purified, and screened for increased thermostability, and 14 of these displayed an apparent increase in melting temperature of .1°C. The two mutations M15L and S23A were combined, and the resulting mFMO variant had a 3°C increase in melting temperature. Adding additional stabilizing single mutations did not further increase thermostability. In line with what we observed with mFMO_20, no major effects were observed on the kinetic parameters of the mFMO M15L/S23A variant. FRESCO has also been used to stabilize the Rhodococcus sp. strain HI-31 cyclohexanone monooxygenase (23), which also belongs to the FMO family. In this case, half of the 128 screened single-mutant variants had modest stabilizing effects. These were combined, using a shuffled library design strategy, into a variant carrying 8 mutations (M8B), which increased the unfolding temperature by 13°C. In contrast to the PROSS mFMO mutant variants, the FRESCO-derived mutations did not appear to form new salt bridges in either M8B or mFMO M15L/S23A. We engineered mFMO to make it more suitable for industrial applications like removing TMA in salmon protein hydrolysates. The mFMO_20 variant was selected as the best candidate due to it being the most thermostable variant of the seven designs. In addition, the optimal pH for mFMO_20, pH 8.0, was slightly lower than that of the native mFMO, pH 8.5, which may also confer an advantage in industrial applications (e.g., the pH of the salmon protein hydrolysate was 6.1). In fact, the optimal pH for all 7 mutant variants was between 7.5 and 8.0 (Table 2), but with no discernible correlation with changes in charge or pI. The optimal temperature of mFMO_20 did not increase compared to that of native mFMO. Still, this minor disadvantage of mFMO_20 was clearly outweighed by the beneficial properties of increased stability when it was tested for its ability to convert TMA to TMAO in the salmon protein hydrolysate (Fig. 3). At both 50°C and 65°C, mFMO_20 was superior to native mFMO in removing TMA, eliminating 95% of TMA, and it also appeared to perform best at 30°C. These results demonstrate that mFMO_20 is indeed more suitable for industrial applications than the native mFMO. However, there are still important hurdles that must be overcome before this Tmm enzyme can be incorporated into an industrial process, especially its dependence on the unstable and expensive cofactor NADPH. The fact that we and others have demonstrated that the Tmms can be engineered opens the possibility for cofactor engineering, which can be used to make the enzyme accept more cost-efficient cofactors. An alternative or complementary strategy is to bring down cost by regenerating the cofactor, e.g., by using glucose dehydrogenase (24). The current work has demonstrated that the PROSS method could successfully predict mFMO variants with increased thermostability. We did not observe any apparent improvement in expression or solubility levels, which frequently follow improvements in stability, which may be due to the fact that the native mFMO enzyme is already readily expressed in soluble form. All 7 variants proposed by PROSS showed increased thermostability, with properties comparable to those of engineered FMO enzymes obtained after screening more than 100 single-mutant variants followed by library shuffling or rational engineering (16, 23). The mFMO_20 variant with its improved stability may be applicable for industrial use as is, because it can reduce the majority of TMA present in fish hydrolysates. It can also serve as an excellent starting point for rational engineering to further improve its catalytic efficiency or for cofactor engineering to make it accept more cost-efficient cofactors. MATERIALS AND METHODS Protein stabilization mutagenesis using the PROSS Web server. The Protein Repair One-Stop Shop (PROSS) server (https://pross.weizmann.ac.il/) was used to predict variants of mFMO with increased stability Thermostable Trimethylamine Monooxygenase Applied and Environmental Microbiology June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 9 Downloaded from https://journals.asm.org/journal/aem on 23 November 2025 by 5.67.84.229.