Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

Transcriptomic analysis of polysaccharide utilization loci reveals substrate preferences in ruminal generalists Segatella bryantii TF1-3 and Xylanibacter ruminicola KHP1.

Bacteria of the genera Xylanibacter and Segatella are among the most dominant groups in the rumen microbiota. They are characterized by the ability to utilize different hemicelluloses and pectin of plant cell-wall as well as plant energy storage polysaccharides. The degradation is possible with the use of cell envelope bound multiprotein apparatuses coded in polysaccharide utilization loci (PULs), which have been shown to be substrate specific. The knowledge of PUL presence in rumen Xylanibacter and Segatella based on bioinformatic analyses is already established and transcriptomic and genetic approaches confirmed predicted PULs for a limited number of substrates. In this study, we transcriptomically identified additional different PULs in Xylanibacter ruminicola KHP1 and Segatella bryantii TF1-3. We also identified substrate preferences and found that specific growth rate and extent of growth impacted the choice of substrates preferentially used for degradation. These preferred substrates were used by both strains simultaneously as judged by their PUL upregulation. Lastly, ß-glucan and xyloglucan were used by these strains in the absence of bioinformatically and transcriptomically identifiable PUL systems.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Biochemical Characterization of a Novel Thermostable Ulvan Lyase from Tamlana fucoidanivorans CW2-9.

Ulvan is a complex sulfated polysaccharide extracted from Ulva, and ulvan lyases can degrade ulvan through a ß-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The Km and Vmax of recombinant EPL15085 toward ulvan are 0.80 mg·mL-1 and 11.22 µmol·min -1 mg-1·mL-1, respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.

Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity.

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

Exploration of three Dyadobacter fermentans enzymes uncovers molecular activity determinants in CE15.

Glucuronoyl esterases (GEs) are serine-type hydrolase enzymes belonging to carbohydrate esterase family 15 (CE15), and they play a central role in the reduction of recalcitrance in plant cell walls by cleaving ester linkages between glucuronoxylan and lignin in lignocellulose. Recent studies have suggested that bacterial CE15 enzymes are more heterogeneous in terms of sequence, structure, and substrate preferences than their fungal counterparts. However, the sequence space of bacterial GEs has still not been fully explored, and further studies on diverse enzymes could provide novel insights into new catalysts of biotechnological interest. To expand our knowledge on this family of enzymes, we investigated three unique CE15 members encoded by Dyadobacter fermentans NS114T, a Gram-negative bacterium found endophytically in maize/corn (Zea mays). The enzymes are dissimilar, sharing ≤ 39% sequence identity to each other' and were considerably different in their activities towards synthetic substrates. Combined analysis of their primary sequences and structural predictions aided in establishing hypotheses regarding specificity determinants within CE15, and these were tested using enzyme variants attempting to shift the activity profiles. Together, the results expand our existing knowledge of CE15, shed light into the molecular determinants defining specificity, and support the recent thesis that diverse GEs encoded by a single microorganism may have evolved to fulfil different physiological functions. KEY POINTS: • D. fermentans encodes three CE15 enzymes with diverse sequences and specificities • The Region 2 inserts in bacterial GEs may directly influence enzyme activity • Rational amino acid substitutions improved the poor activity of the DfCE15A enzyme.

Substrate uptake patterns shape niche separation in marine prokaryotic microbiome.

Marine heterotrophic prokaryotes primarily take up ambient substrates using transporters. The patterns of transporters targeting particular substrates shape the ecological role of heterotrophic prokaryotes in marine organic matter cycles. Here, we report a size-fractionated pattern in the expression of prokaryotic transporters throughout the oceanic water column due to taxonomic variations, revealed by a multi-"omics" approach targeting ATP-binding cassette (ABC) transporters and TonB-dependent transporters (TBDTs). Substrate specificity analyses showed that marine SAR11, Rhodobacterales, and Oceanospirillales use ABC transporters to take up organic nitrogenous compounds in the free-living fraction, while Alteromonadales, Bacteroidetes, and Sphingomonadales use TBDTs for carbon-rich organic matter and metal chelates on particles. The expression of transporter proteins also supports distinct lifestyles of deep-sea prokaryotes. Our results suggest that transporter divergency in organic matter assimilation reflects a pronounced niche separation in the prokaryote-mediated organic matter cycles.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

Impact of N-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C-H Activation in the M. oryzae Lipoxygenase.

Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in plants and animals, fungal LOXs are unique in having appended N-linked glycans. Such important post-translational modifications (PTMs) endow proteins with altered structure, stability, and/or function. In this study, we present the structural and functional outcomes of removing or altering these surface carbohydrates on the LOX from the devastating rice blast fungus, M. oryzae, MoLOX. Alteration of the PTMs did notinfluence the active site enzyme-substrate ground state structures as visualized by electron-nuclear double resonance (ENDOR) spectroscopy. However, removal of the eight N-linked glycans by asparagine-to-glutamine mutagenesis nonetheless led to a change in substrate selectivity and an elevated activation energy for the reaction with substrate linoleic acid, as determined by kinetic measurements. Comparative hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of wild-type and Asn-to-Gln MoLOX variants revealed a regionally defined impact on the dynamics of the arched helix that covers the active site. Guided by these HDX results, a single glycan sequon knockout was generated at position 72, and its comparative substrate selectivity from kinetics nearly matched that of the Asn-to-Gln variant. The cumulative data from model glyco-enzyme MoLOX showcase how the presence, alteration, or removal of even a single N-linked glycan can influence the structural integrity and dynamics of the protein that are linked to an enzyme's catalytic proficiency, while indicating that extensive glycosylation protects the enzyme during pathogenesis by protecting it from protease degradation.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase.

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite -2. Furthermore, the protein has a larger ß5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

Alkaliphilic and thermostable lipase production by leaf litter fungus Leptosphaerulina trifolii A SMR-2011.

Fungi that inhabit fire-prone forests have to be adapted to harsh conditions and fungi affiliated to Ascomycota recovered from foliar litter samples were used for bioprospecting of molecules such as enzymes. Agni's fungi isolated from leaf litter, whose spores are capable of tolerating 110 oC were screened for thermostable lipases. One of the isolates, Leptosphaerulina trifolii A SMR-2011 exhibited high positive lipase activity than other isolates while screening through agar plate assay using Tween 20 in the medium. Maximum lipase activity (173.2 U/mg) of L. trifolii was observed at six days of inoculation and decreased thereafter. Among different oils used, the maximum lipase activity was attained by soybean oil (940.1 U/mg) followed by sunflower oil (917.1 U/mg), and then by mustard oil (884.8 U/mg), showing its specificity towards unsaturated fatty acids. Among the various organic nitrogen sources tested, soybean meal showed maximum lipase activity (985.4 U/mg). The partially purified enzyme was active over a wide range of pH from 8 to 12 with a pH optimum of 11.0 (728.1 U/mg) and a temperature range of 60-80 oC with an optimal temperature of 70 oC (779.1 U/mg). The results showed that lipase produced by L. trifolii is alkali stable and retained 85% of its activity at pH 11.0. This enzyme also showed high thermal stability retaining more than 50% of activity when incubated at 60 oC to 90 °C for 2 h. The ions Ca2+ and Mn2+ induced the lipase activity, while Cu2+ and Zn2+ ions lowered the activity compared to control. These results suggests that the leaf litter fungus L. trifolii serves as a potential source for the production of alkali-tolerant and thermostable lipase.

Structure, mechanism, and evolution of the last step in vitamin C biosynthesis.

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function.

Two extracellular α-arabinofuranosidases are required for cereal-derived arabinoxylan metabolism by Bifidobacterium longum subsp. longum.

Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.