Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.     

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).     

Three new Nudix hydrolases from Escherichia coli

Three members of the Nudix (nucleoside diphosphate X) hydrolase superfamily have been cloned from Escherichia coli MG1655 and expressed. The proteins have been purified and identified as enzymes active on nucleoside diphosphate derivatives with the following specificities. Orf141 (yfaO) is a nucleoside triphosphatase preferring pyrimidine deoxynucleoside triphosphates. Orf153 (ymfB) is a nonspecific nucleoside tri- and diphosphatase and atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. Orf191 (yffH) is a highly active GDP-mannose pyrophosphatase. All three enzymes require a divalent cation for activity and are optimally active at alkaline pH, characteristic of the Nudix hydrolase superfamily. The question of whether or not Orf1.9 (wcaH) is a bona fide member of the Nudix hydrolase superfamily is discussed.     

Discovery of Two β-1,2-Mannoside Phosphorylases Showing Different Chain-Length Specificities from Thermoanaerobacter sp. X-514

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man₂, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.     

A cryptic activity in the Nudix hydrolase superfamily

The Nudix hydrolase superfamily is identified by a conserved cassette of 23 amino acids, and it is characterized by its pyrophosphorylytic activity on a wide variety of nucleoside diphosphate derivatives. Of the 13 members of the family in Escherichia coli, only one, Orf180, has not been identified with a substrate, although a host of nucleoside diphosphate compounds has been tested. Several reports have noted a strong similarity in the three‐dimensional structure of the unrelated enzyme, isopentenyl diphosphate isomerase (IDI) to the Nudix structure, and the report that a Nudix enzyme was involved in the synthesis of geraniol, a product of the two substrates of IDI, prompted an investigation of whether the IDI substrates, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DAPP) could be substrates of Orf180. This article demonstrates that Orf180 does have a very low activity on IPP, DAPP, and geranyl pyrophosphate (GPP). However, several of the other Nudix enzymes with established nucleoside diphosphate substrates hydrolyze these compounds at substantial rates. In fact, some Nudix hydrolases have higher activities on IPP, DAPP, and GPP than on their signature nucleoside diphosphate derivatives.     

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

Cloning of Genes Coding for the Three Subunits of Thiocyanate Hydrolase of Thiobacillus thioparus THI 115 and Their Evolutionary Relationships to Nitrile Hydratase

Thiocyanate hydrolase is a newly found enzyme from Thiobacillus thioparus THI 115 that converts thiocyanate to carbonyl sulfide and ammonia (Y. Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa, and H. Kuraishi, J. Biol. Chem. 267:9170–9175, 1992). We have cloned and sequenced the scn genes that encode the three subunits of the enzyme. The scnB, scnA, and scnC genes, arrayed in this order, contained open reading frames encoding sequences of 157, 126, and 243 amino acid residues, respectively, for the β, α, and γ subunits, respectively. Each open reading frame was preceded by a typical Shine-Dalgarno sequence. The deduced amino-terminal peptide sequences for the three subunits were in fair agreement with the chemically determined sequences. The protein molecular mass calculated for each subunit was compatible with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From a computer analysis, thiocyanate hydrolase showed significant homologies to bacterial nitrile hydratases known to convert nitrile to the corresponding amide, which is further hydrolyzed by amidase to form acid and ammonia. The two enzymes were homologous over regions corresponding to almost the entire coding regions of the genes: the β and α subunits of thiocyanate hydrolase were homologous to the amino- and carboxyl-terminal halves of the β subunit of nitrile hydratase, and the γ subunit of thiocyanate hydrolase was homologous to the α subunit of nitrile hydratase. Comparisons of the catalytic properties of the two homologous enzymes support the model for the reaction steps of thiocyanate hydrolase that was previously presented on the basis of biochemical analyses.     

Role of the NC-Loop in Catalytic Activity and Stability in Lipase from Fervidobacterium changbaicum

Flexible NC-loops between the catalytic domain and the cap domain of the α/β hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the α/β hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131–151) in a lipase (FClip1) from thermophilic bacterium Fervidobacterium changbaicum by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131–138) was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139–151) was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic properties, and might present a hot zone for the divergent evolution of α/β hydrolases. Our findings may provide an opportunity for better understanding of the mechanism of divergent evolution in the α/β hydrolase fold superfamily, and may also guide the design of novel biocatalysts for industrial applications.     

Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M₂), mannotriose (M₃), and mannotetraose (M₄) but not mannopentaose (M₅) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (k_cat/K_m) toward mannohexaose (M₆) compared with the endo-β-1,4-mannanase Man5C and notably converted M₆ to M₂, M₃, and M₄, with M₃ being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.     

Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application.     

Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase⁺ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca²⁺ and Mg²⁺) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.     

The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis

In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn²⁺. EPR studies reveal the presence of a Mn²⁺ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn²⁺ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.     

The Structural Differences between a Glycoprotein Specific F-Box Protein Fbs1 and Its Homologous Protein FBG3

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.     

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of β1-β1′-β2-β3-α-β4-β45₁-β45₂-β5 secondary structure elements. MtuSSB NTD includes an additional β-strand (β6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mβ4-β5), wherein only the terminal part of NTD (β4-β5 region possessing L₄₅ loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mβ4-β6 construct obtained by substitution of the region downstream of β5 in mβ4-β5 SSB with the corresponding region (β6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L₄₅ loop and the β6 strand of MtuSSB.     

Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this β7–β8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by β7–β8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.     

Gene Sets for Utilization of Primary and Secondary Nutrition Supplies in the Distal Gut of Endangered Iberian Lynx

Recent studies have indicated the existence of an extensive trans-genomic trans-mural co-metabolism between gut microbes and animal hosts that is diet-, host phylogeny- and provenance-influenced. Here, we analyzed the biodiversity at the level of small subunit rRNA gene sequence and the metabolic composition of 18 Mbp of consensus metagenome sequences and activity characteristics of bacterial intra-cellular extracts, in wild Iberian lynx (Lynx pardinus) fecal samples. Bacterial signatures (14.43% of all of the Firmicutes reads and 6.36% of total reads) related to the uncultured anaerobic commensals Anaeroplasma spp., which are typically found in ovine and bovine rumen, were first identified. The lynx gut was further characterized by an over-representation of ‘presumptive’ aquaporin aqpZ genes and genes encoding ‘active’ lysosomal-like digestive enzymes that are possibly needed to acquire glycerol, sugars and amino acids from glycoproteins, glyco(amino)lipids, glyco(amino)glycans and nucleoside diphosphate sugars. Lynx gut was highly enriched (28% of the total glycosidases) in genes encoding α-amylase and related enzymes, although it exhibited low rate of enzymatic activity indicative of starch degradation. The preponderance of β-xylosidase activity in protein extracts further suggests lynx gut microbes being most active for the metabolism of β-xylose containing plant N-glycans, although β-xylosidases sequences constituted only 1.5% of total glycosidases. These collective and unique bacterial, genetic and enzymatic activity signatures suggest that the wild lynx gut microbiota not only harbors gene sets underpinning sugar uptake from primary animal tissues (with the monotypic dietary profile of the wild lynx consisting of 80–100% wild rabbits) but also for the hydrolysis of prey-derived plant biomass. Although, the present investigation corresponds to a single sample and some of the statements should be considered qualitative, the data most likely suggests a tighter, more coordinated and complex evolutionary and nutritional ecology scenario of carnivore gut microbial communities than has been previously assumed.     

Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition

Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m⁶ ₂A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity–binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.     

Structure and Biosynthesis of Two Exopolysaccharides Produced by Lactobacillus johnsonii FI9785

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1–3)-β-Glcp-(1–5)-β-Galf-(1–6)-α-Glcp-(1–4)-β-Galp-(1–4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.     

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln²⁶³ serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.     

Role of ERO1-α–mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress–induced apoptosis

Endoplasmic reticulum (ER) stress–induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-α (ER oxidase 1 α). In ER-stressed cells, ERO1-α is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-α suppresses apoptosis. IP3-induced calcium release (IICR) is increased during ER stress, and this response is blocked by siRNA-mediated silencing of ERO1-α or IP3R1 and by loss-of-function mutations in Ero1a or Chop. Reconstitution of ERO1-α in Chop^−/− macrophages restores ER stress–induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not Chop^−/− mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ERO1-α–IP3R pathway.