Diversity of sugar-diphospholipid-utilizing glycosyltransferase families.

Peptidoglycan polymerases, enterobacterial common antigen polymerases, O-antigen ligases, and other bacterial polysaccharide polymerases (BP-Pols) are glycosyltransferases (GTs) that build bacterial surface polysaccharides. These integral membrane enzymes share the particularity of using diphospholipid-activated sugars and were previously missing in the carbohydrate-active enzymes database (CAZy; www.cazy.org ). While the first three classes formed well-defined families of similar proteins, the sequences of BP-Pols were so diverse that a single family could not be built. To address this, we developed a new clustering method using a combination of a sequence similarity network and hidden Markov model comparisons. Overall, we have defined 17 new GT families including 14 of BP-Pols. We find that the reaction stereochemistry appears to be conserved in each of the defined BP-Pol families, and that the BP-Pols within the families transfer similar sugars even across Gram-negative and Gram-positive bacteria. Comparison of the new GT families reveals three clans of distantly related families, which also conserve the reaction stereochemistry.

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Three alginate lyases provide a new gut Bacteroides ovatus isolate with the ability to grow on alginate.

Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an endo-acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Whole-genome sequencing of this alginate-utilizing isolate suggested that it is a new Bacteroides ovatus strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The BoPL6 degraded polyG to oligosaccharides of DP 1-3, and BoPL17 released 4,5-unsaturated monouronate from polyM. BoPL38 degraded both alginates, polyM, polyG, and polyMG, in endo-mode; hence, it was assumed to deliver oligosaccharide substrates for BoPL6 and BoPL17, corresponding well with synergistic action on alginate. BoPL17 and BoPL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (α/α)6-barrel + anti-parallel ß-sheet and (α/α)7-barrel folds, distinctive for these PL families. BoPL17 had a more open active site than the two homologous structures. BoPL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium.IMPORTANCEHuman ingestion of sustainable biopolymers calls for insight into their utilization in our gut. Seaweed is one such resource with alginate, a major cell wall component, used as a food hydrocolloid and for encapsulation of pharmaceuticals and probiotics. Knowledge is sparse on the molecular basis for alginate utilization in the gut. We identified a new Bacteroides ovatus strain from human feces that grew on alginate and encoded three alginate lyases in a gene cluster. BoPL6 and BoPL17 show complementary specificity toward guluronate (G) and mannuronate (M) residues, releasing unsaturated oligosaccharides and monouronic acids. BoPL38 produces oligosaccharides degraded by BoPL6 and BoPL17 from both alginates, G-, M-, and MG-substrates. Enzymatic and structural characterization discloses the mode of action and synergistic degradation of alginate by these alginate lyases. Other bacteria were cross-feeding on alginate oligosaccharides produced by an endo-acting alginate lyase. Hence, there is an interdependent community in our guts that can utilize alginate.

New Insight into the Substrate Selectivity of Bovine Milk γ-glutamyl Transferase via Structural and Molecular Dynamics Predictions.

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl-p-nitroanilide (γ-GpNA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.

Conserved unique peptide patterns (CUPP) online platform 2.0: implementation of +1000 JGI fungal genomes.

Carbohydrate-processing enzymes, CAZymes, are classified into families based on sequence and three-dimensional fold. Because many CAZyme families contain members of diverse molecular function (different EC-numbers), sophisticated tools are required to further delineate these enzymes. Such delineation is provided by the peptide-based clustering method CUPP, Conserved Unique Peptide Patterns. CUPP operates synergistically with the CAZy family/subfamily categorizations to allow systematic exploration of CAZymes by defining small protein groups with shared sequence motifs. The updated CUPP library contains 21,930 of such motif groups including 3,842,628 proteins. The new implementation of the CUPP-webserver, https://cupp.info/, now includes all published fungal and algal genomes from the Joint Genome Institute (JGI), genome resources MycoCosm and PhycoCosm, dynamically subdivided into motif groups of CAZymes. This allows users to browse the JGI portals for specific predicted functions or specific protein families from genome sequences. Thus, a genome can be searched for proteins having specific characteristics. All JGI proteins have a hyperlink to a summary page which links to the predicted gene splicing including which regions have RNA support. The new CUPP implementation also includes an update of the annotation algorithm that uses only a fourth of the RAM while enabling multi-threading, providing an annotation speed below 1 ms/protein.

Rate Response of Poly(Ethylene Terephthalate)-Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase.

The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (XC ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of XC on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with XC . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (XC ≈10 %), but this enzyme was extremely sensitive to increased XC , whereas the enzymes LCCICCG , LCC, and DuraPETase had higher tolerance to increases in XC and had activity on PET disks having XC of 24.4 %. Microscopy revealed that the XC -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.

Elucidation of the Molecular Mechanism of Bovine Milk γ-Glutamyltransferase Catalyzed Formation of γ-Glutamyl-Valyl-Glycine.

γ-Glu-Val-Gly (γ-EVG) is a potent kokumi peptide that can be synthesized through the transpeptidase reaction catalyzed by γ-glutamyl transferase from bovine milk (BoGGT). To explore the molecular mechanism between BoGGT and l-glutamine, γ-glutamyl peptides were generated through the transpeptidase reaction catalyzed by BoGGT at various reaction conditions. Quantitation of γ-glutamyl peptides, structure prediction of BoGGT, molecular docking, and molecular dynamic simulations were performed. Membrane-free BoGGT had a higher transpeptidase activity with Val-Gly as an acceptor than membrane BoGGT. The suitable conditions for γ-EVG production using BoGGT were 100 mM Val-Gly, 20 mM Gln, 1.2 U/mL BoGGT, pH 8.5, and 37 °C, and 13.1 mM γ-EVG was produced. The hydrogen bonds are mainly formed between residues from the light subunit of BoGGT (Thr380, Thr398, Ser450, Ser451, Met452, and Gly473) and the l-glutamine donor. NaCl might inhibit the transpeptidase activity by destroying the hydrogen bonds between BoGGT and l-glutamine, thereby increasing the distance between the hydroxyl oxygen atom on Thr380 of BoGGT and the amide carbon atom on l-glutamine.

Fungal feruloyl esterases can catalyze release of diferulic acids from complex arabinoxylan.

Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acids (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.

A new continuous assay for quantitative assessment of enzymatic degradation of poly(ethylene terephthalate) (PET).

Enzymatic degradation of poly(ethylene terephthalate) (PET) has emerged as a promising route for ecofriendly biodegradation of plastic waste. Several discontinuous activity assays have been developed for assessing PET hydrolyzing enzymes, usually involving manual sampling at different time points during the course of the enzymatic reaction. In this work, we present a novel, compartmentalized UV absorbance assay for continuous detection of soluble hydrolysis products released during enzymatic degradation of PET. The methodology is based on removal of the walls separating two diagonally adjacent wells in UV-transparent microplates, to ensure passage of soluble enzymatic hydrolysis products between the two adjacent wells: One well holds an insoluble PET disk of defined dimensions and the other is used for continuous reading of the enzymatic product formation (at 240 nm). The assay was validated by quantifying the rate of mixing of the soluble PET degradation product BHET (bis(2-hydroxyethyl) terephthalate) between the two adjacent wells. The assay validation also involved a simple adjustment for water evaporation during prolonged assays. With this new assay, we determined the kinetic parameters for two PET hydrolases, DuraPETase and LCCICCG, and verified the underlying assumption of steady-state reaction rates. This new continuous assay enables fast exploration and robust kinetic characterization of PET degrading enzymes.

Standardized method for controlled modification of poly (ethylene terephthalate) (PET) crystallinity for assaying PET degrading enzymes.

Poly(ethylene terephthalate) (PET) is a polyester plastic, which is widely used, notably as a material for single-use plastic bottles. Its accumulation in the environment now poses a global pollution threat. A number of enzymes are active on PET providing new options for industrial biorecycling of PET materials. The enzyme activity is strongly affected by the degree of PET crystallinity (XC), and the XC is therefore a relevant factor to consider in enzyme catalyzed PET recycling. Here, we present a new experimental methodology, based on systematic thermal annealing for controlled preparation of PET disks having different XC, to allow systematic quantitative evaluation of the efficiency of PET degrading enzymes at different degrees of PET substrate crystallinity. We discuss the theory of PET crystallinity and compare PET crystallinity data measured by differential scanning calorimetry and attenuated Fourier transform infrared spectroscopy.•This study introduces a simple method for controlling the crystallinity of PET samples via annealing in a heat block.•The present methodology is not limited to the analytical methods included in the methods details.

Influence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET).

This work examines the significance of the degree of crystallinity (XC) of polyethylene terephthalate (PET) and the PET glass transition temperature (Tg) on enzymatic degradation of PET at elevated temperatures using two engineered, thermostable PET degrading enzymes: LCCICCG, a variant of the leaf-branch compost cutinase, and DuraPETase, evolved from the Ideonella sakaiensis PETase. The XC was systematically varied by thermal annealing of PET disks (Ø 6 mm, thickness 1 mm). The XC affected the enzymatic product release rate that essentially ceased at XC 22-27% for the LCCICCG and at XC ∼17% for the DuraPETase. Scanning Electron Microscopy revealed that enzymatic treatment produced cavities on the PET surface when the XC was > 10% but resulted in a smooth surface on amorphous PET (XC ∼10%). The Tg of amorphous PET disks decreased from 75 °C to 60 °C during 24 h pre-soaking in water at 65 °C, while the XC remained unchanged. Enzymatic reaction on pre-soaked disks at 68 °C, i.e. above the Tg, did not affect the enzymatic product release rate catalyzed by LCCICCG. These findings improve the understanding of enzymatic PET degradation and have implications for development of efficient enzymatic PET upcycling processes.

Conserved unique peptide patterns (CUPP) online platform: peptide-based functional annotation of carbohydrate active enzymes.

The CUPP platform includes a web server for functional annotation and sub-grouping of carbohydrate active enzymes (CAZymes) based on a novel peptide-based similarity assessment algorithm, i.e. protein grouping according to Conserved Unique Peptide Patterns (CUPP). This online platform is open to all users and there is no login requirement. The web server allows the user to perform genome-based annotation of carbohydrate active enzymes to CAZy families, CAZy subfamilies, CUPP groups and EC numbers (function) via assessment of peptide-motifs by CUPP. The web server is intended for functional annotation assessment of the CAZy inventory of prokaryotic and eukaryotic organisms from genomic DNA (up to 30MB compressed) or directly from amino acid sequences (up to 10MB compressed). The custom query sequences are assessed using the CUPP annotation algorithm, and the outcome is displayed in interactive summary result pages of CAZymes. The results displayed allow for inspection of members of the individual CUPP groups and include information about experimentally characterized members. The web server and the other resources on the CUPP platform can be accessed from https://cupp.info.

Insights into substrate binding of ferulic acid esterases by arabinose and methyl hydroxycinnamate esters and molecular docking.

Ferulic acid esterases (FAE, EC 3.1.1.73) cleave the arabinose hydroxycinnamate ester in plant hemicellulose and other related substrates. FAE are commonly categorised as type A-D based on catalytic activities towards model, short alkyl chain esters of hydroxycinnamates. However, this system correlates poorly with sequence and structural features of the enzymes. In this study, we investigated the basis of the type A categorisation of an FAE from Aspergillus niger, AnFaeA, by comparing its activity toward methyl and arabinose hydroxycinnamate esters. kcat/Km ratios revealed that AnFaeA hydrolysed arabinose ferulate 1600-fold, and arabinose caffeate 6.5 times more efficiently than their methyl ester counterparts. Furthermore, small docking studies showed that while all substrates adopted a catalytic orientation with requisite proximity to the catalytic serine, methyl caffeate and methyl p-coumarate preferentially formed alternative non-catalytic conformations that were energetically favoured. Arabinose ferulate was unable to adopt the alternative conformation while arabinose caffeate preferred the catalytic orientation. This study demonstrates that use of short alkyl chain hydroxycinnnamate esters can result in activity misclassification. The findings of this study provide a basis for developing a robust classification system for FAE and form the basis of sequence-function relationships for this class.

Biochemical characterization of a halotolerant feruloyl esterase from Actinomyces spp.: refolding and activity following thermal deactivation.

Ferulic acid esterases (FAE, EC. 3.1.1.73) hydrolyse the linkage between hemicellulose and lignin and thus have potential for use in mild enzymatic pretreatment of biomass as an alternative to thermochemical approaches. Here, we report the characterization of a novel FAE (ActOFaeI) obtained from the bacterium, Actinomyces sp. oral which was recombinantly expressed in Escherichia coli BL21 in two forms: with and without its putative signal peptide. The truncated form was found to have <10 % relative activity compared to the full length and was more prone to aggregation after purification. The enzyme with retained peptide demonstrated 2 to 4-fold higher activity against methyl caffeate and methyl p-coumarate, with specific activities of 477.6 and 174.4 U mg(-1) respectively, than the equivalent activities of the benchmark FAE from Aspergillus niger A and B. ActOFaeI retained activity over a broad pH range with a maximum at 9 but >90 % relative activity at pH 6.5 and an optimum reaction temperature of 30 °C. ActOFaeI increased activity by 15% in high salt conditions (1000 mMNaCl) and its thermal unfolding temperature improved from 41.5 °C in standard buffer to 74 °C in the presence of 2500 mM sodium malonate. ActOFaeI also released ferulic acid from destarched wheat bran when combined with a xylanase preparation. After treatment above the thermal denaturation temperature followed by cooling to room temperature, ActOFaeI demonstrated spontaneous refolding into an active state. ActOFaeI displays many useful characteristics for enzymatic pretreatment of lignocellulose and contributes to our understanding of this important family.