Biology subjects

Ree, R.

Publications and source records attributed to Ree, R..

7 recordsLinked to original sources

bioRxiv2026

Multi-objective Engineering of Trimethylamine Monooxygenase for Improved Thermostability and Cofactor Use

Trimethylamine (TMA) is a major contributor to undesirable odours in protein hydrolysates derived from marine by-products, limiting their industrial use. Flavin-containing monooxygenases (FMOs) catalyse the conversion of TMA to the odourless trimethylamine N-oxide (TMAO); however, industrial applications demand enzymes that are both thermally stable and compatible with cost-effective cofactors. A thermostable variant of the Methylophaga aminisulfidivorans FMO (mFMO_20) can function at elevated temperatures but depends exclusively on the expensive and unstable cofactor NADPH. In this study, we investigated whether it is possible to simultaneously enhance thermostability and NADH compatibility using a multi-objective engineering strategy. We first targeted residues in the cofactor binding site of mFMO_20 to restore NADH activity, which had been completely lost despite the wild type enzyme being naturally active with both cofactors. Variants derived from the thermostable scaffold partially recovered NADH activity but showed reduced NADPH activity. Given the wild types inherent NADH compatibility, we next pursued a stability-improvement approach, introducing highly conserved stabilizing mutations. This preserved cofactor competence but produced only modest improvements in thermostability. Finally, by combining physical, evolutionary, and statistical metrics, we obtained variants that retained higher NADPH activity after heat treatment than any previously reported thermostable mutants, while a subset also retained measurable NADH activity before heat treatment. These findings show that combining complementary scoring strategies helps navigate the trade-off between stability and activity; while, robust NADH function under thermal stress remains elusive, with only one variant retaining detectable NADH activity after heat treatment, the results provide valuable insight into the underlying constraints linking stability and cofactor usage and highlights possible directions for engineering FMOs with both enhanced thermostability and cofactor compatibility. Author summaryIn this work, we aimed to improve an enzyme that could be useful in industrial applications but is limited by two common constraints: poor stability at high temperatures and dependence on an expensive cofactor. To make the enzyme more suitable for large-scale applications, we sought to engineer variants that are both more thermostable and compatible with a cheaper cofactor, NADH. For enzyme engineering, we used a strategy that balances several properties rather than prioritizing a single trait. We combined tools that capture evolutionary patterns, protein physics, and AI-based predictions to explore which mutations might provide the right combination of stability and function. Through this approach, we obtained variants with improved heat resistance and higher cofactor activity retention.

molecular biology↗

bioRxiv2025

Monooxygenase-dehydrogenase cascade for sustained enzymatic remediation of TMA in salmon protein hydrolysates

Fish protein hydrolysates hold great promise as nutraceuticals, yet their application as food ingredients or nutraceuticals is currently limited by their fish-like odor. This odor is mainly due to the presence of trimethylamine (TMA), a volatile biogenic amine resulting from the breakdown of naturally occurring trimethylamine-N-oxide (TMAO) in marine fish. The bacterial trimethylamine monooxygenase mFMO can oxidize TMA into TMAO using molecular oxygen and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). We have established an enzyme cascade which takes advantage of glucose dehydrogenase to recycle NADPH from NADP+, significantly decreasing the cost of the reaction and paving the way for using the enzyme system in fish protein hydrolysates targeted for human consumption. We demonstrate that the dual enzyme system works in an industrially relevant substrate. Salmon protein hydrolysate treated with an mFMO/glucose dehydrogenase cocktail showed a 75% reduction in TMA. A trained sensory panel perceived an improved odor across several parameters, including a reduction in the characteristic TMA smell.

biochemistry↗

bioRxiv2024

Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish

Actin is a key component of the cytoskeleton of eukaryotic cells and is involved in numerous cellular functions. In animal cells, actins are uniquely N-terminally processed by a dedicated enzyme machinery to generate their mature acidic and acetylated forms. The final step of this maturation process involves N-terminal acetylation, a reaction catalyzed by NAA80 in humans. In human cell lines, N-terminal acetylation of actin plays a crucial role in maintaining normal cytoskeletal dynamics and cell motility. The physiological impact of actin N-terminal acetylation remains to be defined. Here, we developed a zebrafish naa80 knockout model and established that zNaa80 acetylates both muscle and non-muscle actins in vivo. Our in vitro investigation of purified zNaa80 unveiled a clear preference for acetylating N-termini derived from actins. Interestingly, zebrafish lacking actin N-terminal acetylation were viable and exhibited normal development, morphology and behaviour. In contrast, human individuals carrying pathogenic actin variants may present with hypotonia and hearing impairment. While zebrafish depleted for naa80 did not display any obvious muscle defects or abnormal muscle tissue, we found that they have abnormal inner ear development such as small otoliths and impaired response to sound stimuli. In sum, we have defined that zebrafish Naa80 N-terminally acetylates actins in vitro and in vivo and that actin N-terminal acetylation is essential for normal hearing in vivo.

biochemistry↗

bioRxiv2023

Increased thermostability of an engineered flavin-containing monooxygenase to remediate trimethylamine in fish protein hydrolysates

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 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 8-28 mutations, displayed increases in melting temperature between 4.7 {degrees}C and 9.0 {degrees}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. ImportanceMarine by-products are a high-quality source for peptide ingredients, but the unpleasant fishy odour caused by TMA limits their access to the food market. This problem can be mitigated by enzymatic conversion of TMA into the odourless TMAO. Enzymes isolated from nature must be adapted to industrial requirements, however, 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 towards application of this novel and highly promising enzyme technology in marine biorefineries.

molecular biology↗

bioRxiv2022

Loss of N-terminal acetyltransferase A activity induces thermally unstable ribosomal proteins and increases their turnover

Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering [~]50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employed a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which caused global proteome remodeling. Pulsed-SILAC experiments revealed that NatA-deficient strains consistently increased degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovered decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding. TeaserA multidimensional proteomics approach reveals the effect of N-terminal acetylation on Saccharomyces cerevisiae cytosolic ribosomal proteins.

biochemistry↗

bioRxiv2020

Naa12 rescues embryonic lethality in Naa10-Deficient Mice in the amino-terminal acetylation pathway

Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralogue with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

developmental biology↗

bioRxiv2020

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. Here, we define a specific role for PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80. PFN2 binding increases the intrinsic catalytic activity of NAA80. Furthermore, binding of PFN2 to NAA80 via its proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. The majority of NAA80 is stably bound to PFN2, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation. Data are available via ProteomeXchange with identifier PXD020188.

biochemistry↗