Biology subjects

Aachmann, F. L.

Publications and source records attributed to Aachmann, F. L..

10 recordsLinked to original sources

bioRxiv2024

The Swiss Army Knife of Alginate Manipulation - A Gut Bacterium Alginate Lyase with Diverse Catalytic Activities

The alginate-degrading enzyme BoPL38 of the human gut bacterium Bacteroides ovatus CP926 degrades the three polysaccharide structures found in alginate, a major constituent of brown macroalgae with numerous industrial applications. However, the detailed mechanisms of alginate-degrading enzymes remain unclear. Crystal structures of BoPL38 complexes with alginate oligosaccharides, now shed light on the enzymes catalytic machinery. QM/MM simulations reveal distinct conformational and reaction pathways, highlighting different transition states for mannuronate and guluronate conversion. C5 proton abstraction at subsite +1 by Y298 and H243 facilitates syn- and anti-{beta}-elimination reactions, respectively. Substrate recognition relies on R292 distorting the sugar at subsite +1 into a preactivated conformation, while stabilizing the active site tunnel through a salt bridge. Furthermore, NMR spectroscopy found that BoPL38 also catalyze mannuronate to guluronate epimerization in addition to its lyase function, thereby paving the way for future enzymatic alginate modification.

biochemistry↗

bioRxiv2024

Innovations in Alginate Catabolism Leading to Heterotrophy and Adaptive Evolution of Diatoms

A major goal of evolutionary biology is to identify the genetic basis for the emergence of adaptive traits. Diatoms are ancestrally photosynthetic microalgae. However, in the genus Nitzschia, loss of photosynthesis led to a group of free-living secondary heterotrophs whose manner of energy acquisition is unclear. Here, we sequence the genome of the non-photosynthetic diatom Nitzschia sing1 and identify the genetic basis for its catabolism of the brown seaweed cell wall polysaccharide alginate. N. sing1 obtained an endolytic alginate lyase enzyme by horizontal gene transfer (HGT) from a marine bacterium. Subsequent gene duplication and transposition led to 91 genes in three distinct gene families. One family retains the ancestral endolytic enzyme function. By contrast, the two others underwent domain duplication, gain, loss, rearrangement, and mutation to encode novel functions that can account for oligosaccharide import through the endomembrane system and the exolytic production of alginate monosaccharides. Together, our results show how a single HGT event followed by substantial gene duplication and neofunctionalization led to alginate catabolism and access to a new ecological niche. HighlightsO_LIN. sing1 acquired an alginate lyase (ALY) gene by horizontal gene transfer from a marine bacterium C_LIO_LIThis founding gene expanded and diversified to comprise 3 major families across 30 loci C_LIO_LIDerived functions account for alginate import and processing into monomers C_LIO_LIDomain duplication, gain, loss, mutation, and de novo sequence evolution underlie ALY gene neofunctionalization C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/610029v1_ufig1.gif" ALT="Figure 1000"> View larger version (44K): org.highwire.dtl.DTLVardef@7a47c7org.highwire.dtl.DTLVardef@1fe958forg.highwire.dtl.DTLVardef@10ce362org.highwire.dtl.DTLVardef@12465a1_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

evolutionary biology↗

bioRxiv2024

Phosphate starvation stops bacteria digesting algal fucan that sequesters carbon

Algae provide a solar powered pathway to capture and sequester carbon by injecting stable fucan made from carbon dioxide into the ocean 1-4. Stability of the pathway is at odds with the presence of marine bacteria with genes of enzymes that can digest fucan and release the carbon dioxide 5. Biochemical explanations for stable fucan remain hypothetical 6. We assembled a biological carbon cycle model and found phosphate limitation enhanced fucan synthesis by algae, stopped digestion by bacteria and thereby stabilized the fucan carbon sequestration pathway. Marine microalgae Glossomastix sp. PLY432 increased synthesis of fucan, a part of its extracellular matrix, under nutrient-growth limiting conditions. Rate and extent of fucan digestion by a marine, isolated bacterium of the Akkermansiaceae family decreased with decreasing phosphate concentration. Phosphate starvation restricted bacterial growth rate, biomass yield and in turn increased the amount of stable fucan. Phosphate is universally required for growth but rare relative to glycan carbon in photosynthesis-derived ecosystems. The fact that phosphate is required for replication, transcription and translation explains why bacteria can digest gigatons of laminarin with a few enzymes, but not fucan during nutrient limited algal blooms. We conclude phosphate starvation constrains the ability of bacteria to digest fucan, which evolves to maintain stability around algal cells and consequentially also to keep carbon dioxide in the ocean.

microbiology↗

bioRxiv2023

Experimental and computational insights into the functional and structural role of the linker region of a two-domain lytic polysaccharide monooxygenase

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the wild-type enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using SAXS, NMR, MD simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the wild-type and a serine-rich variant, which have the same linker length, demonstrated better performance compared to those with either a shorter or longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, while the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass processing enzymes is more complex than mere structural tethering.

biochemistry↗

bioRxiv2023

Following the fate of lytic polysaccharide monooxygenases (LPMOs) under oxidative conditions by NMR spectroscopy

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO action is key to the efficient varlorization of biomass, but the instability of LPMOs in turnover conditions limits their efficiency. LPMO catalysis requires the presence of a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors.. While it is known that reduced LPMOs are prone to auto-catalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage. Here, we present atomic-level insight into how the structure of the chitin-active SmLPMO10A is affected by oxidative damage, using NMR and CD spectroscopy. Incubation with ascorbic acid, led to rearrangements of aromatic residues, followed by more profound structural changes near the copper active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate. The impact of turnover conditions on aromatic residues in the core of the enzyme suggests a role for these residues in dealing with redox-active species generated in the copper center.

biochemistry↗

bioRxiv2021

NMR structures and functional roles of two related chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. Still, structural and functional properties of some of these CBMs remain unknown and it is not clear why some LPMOs, like CjLPMO10A from Cellvibrio japonicus, have two CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on the functional variation, and determined the solution structures of both by NMR, which includes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the {micro}M range, CjCBM73 has higher affinity than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding to soluble chitin was detected for CjCBM73. These functional differences correlated with distinctly different architectures of the substrate-binding surfaces of the two CBMs. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

biochemistry↗

bioRxiv2021

A mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing {beta}-D-mannuronic acid residues (M) into -L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through {beta}-elimination. The reaction mechanisms for the epimerization and cleavage are similar and some enzymes can perform both. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their activity as well as the amino acids responsible for it are still unknown. In this study, we investigate mechanistic determinants of the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that the lyase activity of AlgE7 is regulated by the type of ion present: Calcium promotes it, whereas NaCl reduces it. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M{downarrow}XM and G{downarrow}XM, where X can be either M or G. By studying AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 as the catalytic acid. ImportancePost-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine and material industries. By providing a better understanding of the reaction mechanism and of how the two enzyme reactions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

biochemistry↗

bioRxiv2020

Protective face mask filter capable of inactivating SARS-CoV-2, and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating neither SARS-CoV-2 nor multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2 mediated pneumonia disease complex and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a novel protective non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating SARS-CoV-2 in one minute of contact, and the life-threatening methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many health care workers and researchers working in this urgent and challenging field.

bioengineering↗

bioRxiv2020

Synthesis of Glycoconjugates Utilizing the Regioselectivity of a Lytic Polysaccharide Monooxygenase

Polysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.

biochemistry↗

bioRxiv2019

A pair of esterases from a commensal gut bacterium remove acetylations from all positions on complex β-mannans

{beta}-Mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. {beta}-Mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs), however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, RiCE2 and RiCEX, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The 3D-structure of RiCEX with a mannopentaose in the active site shows that the CBM35 domain of RiCEX forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points towards the Ser41 of the catalytic triad. Cavities on the RiCEX surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In depth characterization of the two enzymes using time-resolved NMR, HPLC and mass spectrometry demonstrates that they work in a complementary manner. RiCEX exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O- and 6-O-acetylations. Activity of RiCE2 is dependent on RiCEX removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O specific RiCEX provided new insight to how temperature and pH affects acetyl migration on mannooligosaccharides.\n\nSignificance statementAcetylations are an important feature of hemicellulose, altering the physical properties of the plant cell wall, and limiting enzyme accessibility. Removal of acetyl groups from beta-mannan is a key step towards efficient utilization of mannans as a carbon source for gut microbiota and in biorefineries. We present detailed insight into mannan deacetylation by two highly substrate-specific acetyl-mannan esterases (AcMEs) from a prevalent gut commensal Firmicute, which cooperatively deacetylate complex galactoglucomannan. The 3D structure of RiCEX with mannopentaose in the active site has a unique two-domain architecture including a CBM35 and an SGNH superfamily hydrolytic domain. Discovery of mannan specific esterases improves the understanding of an important step in dietary fiber utilization by gut commensal Firmicutes.

biochemistry↗