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Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly) 10 , suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2' positions, which may be attributed to the larger space available for substrate binding at the S2 and S2' sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Gram-negative Sphingomonas sp. A1 incorporates acidic polysaccharide alginate into the cytoplasm via a cell-surface alginate-binding protein (AlgQ2)-dependent ATP-binding cassette transporter (AlgM1M2SS). We investigated the function of calcium bound to the EF-hand-like motif in AlgQ2 by introducing mutations at the calcium-binding site. The X-ray crystallography of the AlgQ2 mutant (D179A/E180A) demonstrated the absence of calcium binding and significant disorder of the EF-hand-like motif. Distinct from the wild-type AlgQ2, the mutant was quite unstable at temperature of strain A1 growth, although unsaturated alginate oligosaccharides stabilized the mutant by formation of substrate/protein complex. In the assay of ATPase and alginate transport by AlgM1M2SS reconstructed in the liposome, the wild-type and mutant AlgQ2 induced AlgM1M2SS ATPase activity in the presence of unsaturated alginate tetrasaccharide. These results indicate that the calcium bound to EF-hand-like motif stabilizes the substrate-unbound AlgQ2 but is not required for the complexation of substrate-bound AlgQ2 and AlgM1M2SS., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis , which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities.

Klebsiella pneumoniae pullulanase (KPP) belongs to glycoside hydrolase family 13 subfamily 13 (GH13_13) and is the only enzyme that is reported to perform an induced-fit motion of the active-site loop (residues 706-710). Comparison of pullulanase structures indicated that only KPP has Leu680 present behind the loop, in contrast to the glycine found in other GH13_13 members. Analysis of the structure and activity of recombinant pullulanase from K. pneumoniae ATCC 9621 (rKPP) and its mutant (rKPP-G680L) indicated that the side chain of residue 680 is important for the induced-fit motion of the loop 706-710 and alters the binding affinity of the substrate.

Crystal structures of Klebsiella pneumoniae pullulanase (KPP) in complex with α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) were refined at around 1.98-2.59 Å resolution from data collected at SPring-8. In the structures of the complexes obtained with 1 mM α-CD or γ-CD, one molecule of CD was found at carbohydrate-binding module 41 only (CBM41). In the structures of the complexes obtained with 1 mM β-CD or with 10 mM α-CD or γ-CD, two molecules of CD were found at CBM41 and in the active-site cleft, where the hydrophobic residue of Phe746 occupies the inside cavity of the CD rings. In contrast to α-CD and γ-CD, one β-CD molecule was found at the active site only in the presence of 0.1 mM β-CD. These results were coincident with the solution experiments, which showed that β-CD inhibits this enzyme more than a thousand times more potently than α-CD and γ-CD. The strong inhibition of β-CD is caused by the optimized interaction between β-CD and the side chain of Phe746. The increased K i values of the F746A mutant for β-CD supported the importance of Phe746 in the strong interaction of pullulanase with β-CD.

Endo-1,3-β-glucanase from Cellulosimicrobium cellulans is composed of a catalytic domain and a carbohydrate-binding module. We have determined the X-ray crystal structure of the catalytic domain at a high resolution of 1.66Å. The overall fold is a sandwich-like β-jelly roll architecture like the enzymes in the glycoside hydrolase family 16. The substrate-binding cleft has a length and a width of ~28 and ~15Å, respectively, which is thought to be capable of accommodating at least six glucopyranose units. Laminarihexaose was placed into the substrate-binding cleft, namely at the subsites +2 to -4 from the reducing end, and the complex structure was analyzed using molecular dynamics simulations (MD) and using a rotamer search of the pocket. During the MD simulations, the substrate fluctuated more than the enzyme, where the residues at the subsites toward the non-reducing end fluctuated more than those toward the reducing end. Little conformational change of the protein was observed for the subsites +1 and +2, indicating that the glucose's position could be tightly restricted inside the pocket. Substrate binding experiments using isothermal titration calorimetry showed that the binding affinity of laminaritriose was higher than that of laminaribiose and similar to those of other longer laminarioligosaccharides. Taken together, the substrates mainly bind to the subsites -1 to -3 with the highest affinity, while the part bound to the reducing end would be hydrolyzed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis, the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by polysaccharide lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N-acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.

The tripartite EfeUOB system functions as a low pH iron importer in Gram-negative bacteria. In the alginate-assimilating bacterium Sphingomonas sp. strain A1, an additional EfeO-like protein (Algp7) is encoded downstream of the efeUOB operon. Here we show the metal binding mode of Algp7, which carries a M_75 metallopeptidase motif. The Algp7 protein was purified from recombinant E. coli cells and was subsequently characterized using differential scanning fluorimetry, fluorescence spectrometry, atomic absorption spectroscopy, and X-ray crystallography. The fluorescence of a dye, SYPRO Orange, bound to denatured Algp7 in the absence and presence of metal ions was measured during heat treatment. The fluorescence profile of Algp7 in the presence of metals such as ferric, ferrous, and zinc ions, shifted to a higher temperature, suggesting that Algp7 binds these metal ions and that metal ion-bound Algp7 is more thermally stable than the ligand-free form. Algp7 was directly demonstrated to show an ability to bind copper ion by atomic absorption spectroscopy. Crystal structure of metal ion-bound Algp7 revealed that the metal ion is bound to the cleft surrounded by several acidic residues. Four residues, Glu79, Glu82, Asp96, and Glu178, distinct from the M_75 motif (His115xxGlu118), are coordinated to the metal ion. This is the first report to provide structural insights into metal binding by the bacterial EfeO element., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

FtsZ, a tubulin homologue, is an essential protein of the Z-ring assembly in bacterial cell division. It consists of two domains, the N-terminal and C-terminal core domains, and has a conserved C-terminal tail region. Lateral interactions between FtsZ protofilaments and several Z-ring associated proteins (Zaps) are necessary for modulating Z-ring formation. ZapD, one of the positive regulators of Z-ring assembly, directly binds to the C-terminal tail of FtsZ and promotes stable Z-ring formation during cytokinesis. To gain structural and functional insights into how ZapD interacts with the C-terminal tail of FtsZ, we solved two crystal structures of ZapD proteins from Salmonella typhimurium (StZapD) and Escherichia coli (EcZapD) at a 2.6 and 3.1 Å resolution, respectively. Several conserved residues are clustered on the concave sides of the StZapD and EcZapD dimers, the suggested FtsZ binding site. Modeled structures of EcZapD-EcFtsZ and subsequent binding studies using bio-layer interferometry also identified the EcFtsZ binding site on EcZapD. The structural insights and the results of bio-layer interferometry assays suggest that the two FtsZ binding sites of ZapD dimer might be responsible for the binding of ZapD dimer to two protofilaments to hold them together.

Oligopeptide-binding proteins (Opps) are part of the ATP-binding cassette system, playing a crucial role in nutrient uptake and sensing the external environment in bacteria, including hyperthermophiles. Opps serve as a binding platform for diverse peptides; however, how these peptides are recognized by Opps is still largely unknown and few crystal structures of Opps from hyperthermophiles have been determined. To facilitate such an understanding, the crystal structure of a putative Opp, OppA from Thermotoga maritima (TmOppA), was solved at 2.6-Å resolution in the open conformation. TmOppA is composed of three domains. The N-terminal domain consists of twelve strands, nine helices, and four 310 helices, and the C-terminal domain consists of five strands, ten helices, and one 310 helix. These two domains are connected by the linker domain, which consists of two strands, three helices, and three 310 helices. Based on structural comparisons of TmOppA with other OppAs and binding studies, we suggest that TmOppA might be a periplasmic Opp. The most distinct feature of TmOppA is the insertion of two helices, which are lacking in other OppAs. A cavity volume between the N-terminal and C-terminal domains is suggested to be responsible for binding peptides of various lengths.

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

Spo0M is a sporulation-control protein that is thought to play an essential role in the early stage of endospore formation. While little is known about the functions of Spo0M, a recent phylogenetic study suggests that, based on its amino-acid sequence, Spo0M might belong to the arrestin clan. The crystal structure of the Spo0M protein was determined at a resolution of 2.3 Å. Ten amino acids at the end of the N-terminus were removed to improve the thermal stability of the purified Spo0M protein and the crystal structure of Spo0M was determined by SAD. Spo0M has a well conserved N-terminal domain with an arrestin-like fold, which consists of a β-strand sandwich structure. Surprisingly, the C-terminal domain of Spo0M, which has no structural homology to arrestin-clan proteins, bears significant structural similarity to the FP domain of the human PI31 protein. In addition, Spo0M harbours a potential polar-core structure connecting the N- and C-terminal domains with several salt bridges, as seen in the crystal structures of arrestin and VPS26. The structure reported here constitutes the first structural information on a bacterial protein that shares significant structural homology to members of the arrestin clan and the FP domain.

5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD(+) and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPCDH are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active site residues in the reaction mechanism are discussed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharides to monosaccharides through four alginate lyase reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-D-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R', with a preference for NADH. Here, we show the identification of a novel NADH-dependent DEH reductase A1-R' in strain A1, structural determination of A1-R' by x-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R'. A1-R' was purified from strain A1 cells and enzymatically characterized. Except for the coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R'. Crystal structures of A1-R' and A1-R'·NAD(+) complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R' and A1-R were similar, although a difference in the coenzyme-binding site (particularly the nucleoside ribose 2' region) was observed. Distinct from A1-R, A1-R' included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R' mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pedobacter heparinus (formerly known as Flavobacterium heparinum) is a typical glycosaminoglycan-degrading bacterium that produces three heparin lyases, Hep I, Hep II, and Hep III, which act on heparins with 1,4-glycoside bonds between uronate and amino sugar residues. Being different from Hep I and Hep II, Hep III is specific for heparan sulfate. Here we describe the crystal structure of Hep III with the active site located in a deep cleft. The X-ray crystallographic structure of Hep III was determined at 2.20 Å resolution using single-wavelength anomalous diffraction. This enzyme comprised an N-terminal α/α-barrel domain and a C-terminal antiparallel β-sheet domain as its basic scaffold. Overall structures of Hep II and Hep III were similar, although Hep III exhibited an open form compared with the closed form of Hep II. Superimposition of Hep III and heparin tetrasaccharide-bound Hep II suggested that an active site of Hep III was located in the deep cleft at the interface between its two domains. Three mutants (N240A, Y294F, and H424A) with mutations at the active site had significantly reduced enzyme activity. This is the first report of the structure-function relationship of P. heparinus Hep III.

Alginate is a heteropolysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substrate-bound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.

The flagellar protein FlgJ, a member of glycoside hydrolase family 73, has N- and C-terminal domains that are responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. The crystal structure of the C-terminal domain of SPH1045 (SPH1045-C), the FlgJ from Sphingomonas sp. strain A1, showed a long cleft formed by two lobes, alpha and beta. In this study, seven site-specific mutants of residues in the cleft were prepared and analyzed. Enzyme activity was reduced most significantly in mutants E185A and Y281A, followed by E224A. A comparison of the crystal structure of the inactive mutant E185A with that of other related enzymes revealed that Glu185 is structurally reasonable as the proton donor and that Tyr281 is close to Glu185. Glu224 is, however, far from the catalytic site, which is inconsistent with the decreased activity exhibited by E224A. The structural flexibility of Glu224 and its neighboring residues observed in SPH1045-C may indicate that this region is able to change its conformation upon substrate binding., (Copyright 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Sphingomonas sp. A1, a Gram-negative bacterium, directly internalizes alginate macromolecules through a mouth-like pit that is present on its cell surface. The alginate-binding protein Algp7, which was found to be localized on the cell surface, contributes to the accumulation of alginate in the pit. Algp7 was crystallized at 293 K by means of the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as a crystallizing agent. Preliminary X-ray analysis showed that the Algp7 crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.1, b = 98.0, c = 100.1 A, and that it diffracted to 2.8 A resolution.

Sphingomonas sp. A1 alginate lyase A1-II', a member of polysaccharide lyase family 7, shows a broad substrate specificity acting on poly alpha-L-guluronate (poly(G)), poly beta-D-mannuronate (poly(M)) and the heteropolymer (poly(MG)) in alginate molecules. A1-II' with a glove-like beta-sandwich as a basic scaffold forms a cleft covered with two lid loops (L1 and L2). Here, we demonstrate the loop flexibility for substrate binding and structural determinants for broad substrate recognition and catalytic reaction. The two loops associate mutually over the cleft through the formation of a hydrogen bond between their edges (Asn141 and Asn199). A double mutant, A1-II' N141C/N199C, has a disulfide bond between Cys141 and Cys199, and shows little enzyme activity. Adding dithiothreitol to the enzyme reaction mixture leads to a tenfold increase in its molecular activity, suggesting the significance of flexibility in lid loops for accommodating the substrate into the active cleft. In alginate trisaccharide (GGG or MMG)-bound A1-II' Y284F, the enzyme interacts appropriately with substrate hydroxyl groups at subsites +1 and +2 and accommodates G or M, while substrate carboxyl groups are strictly recognized by specific residues. This mechanism for substrate recognition enables A1-II' to show the broad substrate specificity. The structure of A1-II' H191N/Y284F complexed with a tetrasaccharide bound at subsites -1 to +3 suggests that Gln189 functions as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid. This is, to our knowledge, the first report on the structure and function relationship in family 7.

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-D-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an alpha6/alpha6-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (D-mannose) at 1.6 A resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, beta-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, alpha6/alpha6-barrel.

Rhamnogalacturonan (RG) lyase produced by plant pathogenic and saprophytic microbes plays an important role in degrading plant cell walls. An extracellular RG lyase YesW from saprophytic Bacillus subtilis is a member of polysaccharide lyase family 11 and cleaves glycoside bonds in polygalacturonan as well as RG type-I through a beta-elimination reaction. Crystal structures of YesW and its complex with galacturonan disaccharide, a reaction product analogue, were determined at 1.4 and 2.5 A resolutions with final R-factors of 16.4% and 16.6%, respectively. The enzyme is composed of an eight-bladed beta-propeller with a deep cleft in the center as a basic scaffold, and its structural fold has not been seen in polysaccharide lyases analyzed thus far. Structural analysis of the disaccharide-bound YesW and a site-directed mutagenesis study suggested that Arg-452 and Lys-535 stabilize the carboxyl group of the acidic polysaccharide molecule and Tyr-595 makes a stack interaction with the sugar pyranose ring. In addition to amino acid residues binding to the disaccharide, one calcium ion, which is coordinated by Asp-401, Glu-422, His-363, and His-399, may mediate the enzyme activity. This is, to our knowledge, the first report of a new structural category with a beta-propeller fold in polysaccharide lyases and provides structural insights into substrate binding by RG lyase.

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.

A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.

Generally, when microbes assimilate macromolecules, they incorporate low-molecular-weight products derived from macromolecules through the actions of extracellular degrading enzymes. However, a Gram-negative bacterium, Sphingomonas sp. A1, has a smart biosystem for the import and depolymerization of macromolecules. The bacterial cells directly incorporate a macromolecule, alginate, into the cytoplasm through a "superchannel", as we named it. The superchannel consists of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Cytoplasmic polysaccharide lyases depolymerize alginate into the constituent monosaccharides. Other than the proteins characterized so far, novel proteins (e.g., flagellin homologs) have been found to be crucial for the import and depolymerization of alginate through genomics- and proteomics-based identification, thus indicating that the biosystem is precisely constructed and regulated by diverse proteins. In this review, we focus on the structure and function of the bacterial biosystem together with the evolution of related proteins.

Glucose kinase, which we tentatively use in this review, represents the enzymes catalyzing the phosphorylation of glucose and other hexoses by means of phosphoryl donors (ATP, ADP, and inorganic polyphosphate [poly(P)]). Except for glucose kinases utilizing ADP, all other glucose kinases belong to the hexokinase (HK) family and are classified into three groups based on primary structural information, i.e., groups HK, A, and B. The structural and evolutionary relationships of glucose kinases belonging to the above three groups have been controversial due to the lack of tertiary structural information on those in groups A and B. However, recent studies on the tertiary structures of poly(P)/ATP-glucomannokinase (GMK: a glucose kinase in group B) from Arthrobacter sp. strain KM and glucokinase (GK) (ecoGK: a glucose kinase in group A) from Escherichia coli have shed light on this problem. A comparison of the tertiary structures of GMK and ecoGK with those of glucose kinases in group HK demonstrated that both GMK and ecoGK are structurally homologous with glucose kinases in group HK, and that glucose kinases belonging to groups HK, A, and B in the HK family evolved divergently from a common ancestor. Based on the simple structure of GMK compared to those of ecoGK and glucose kinases in group HK, and the putative poly(P)-binding site in GMK, we propose that the ancestor of glucose kinases in the HK family was similar to GMK and used poly(P). We also discuss the ancestor and evolutionary process of ROK proteins, whose primary structures are homologous with those of glucose kinases in group B, in connection with the ancestor and evolutionary process of glucose kinases in the HK family.

The optimum pH of Bacillus cereus beta-amylase (BCB, pH 6.7) differs from that of soybean beta-amylase (SBA, pH 5.4) due to the substitution of a few amino acid residues near the catalytic base residue (Glu 380 in SBA and Glu 367 in BCB). To explore the mechanism for controlling the optimum pH of beta-amylase, five mutants of BCB (Y164E, Y164F, Y164H, Y164Q, and Y164Q/T47M/Y164E/T328N) were constructed and characterized with respect to enzymatic properties and X-ray structural crystal analysis. The optimum pH of the four single mutants shifted to 4.2-4.8, approximately 2 pH units and approximately 1 pH unit lower than those of BCB and SBA, respectively, and their k(cat) values decreased to 41-3% of that of the wild-type enzyme. The X-ray crystal analysis of the enzyme-maltose complexes showed that Glu 367 of the wild type is surrounded by two water molecules (W1 and W2) that are not found in SBA. W1 is hydrogen-bonded to both side chains of Glu 367 and Tyr 164. The mutation of Tyr 164 to Glu and Phe resulted in the disruption of the hydrogen bond between Tyr 164 Oeta and W1 and the introduction of two additional water molecules near position 164. In contrast, the triple mutant of BCB with a slightly decreased pH optimum at pH 6.0 has no water molecules (W1 and W2) around Glu 367. These results suggested that a water-mediated hydrogen bond network (Glu 367...W1...Tyr 164...Thr 328) is the primary requisite for the increased pH optimum of wild-type BCB. This strategy is completely different from that of SBA, in which a hydrogen bond network (Glu 380...Thr 340...Glu 178) reduces the optimum pH in a hydrophobic environment.

Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a beta-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 degrees C. The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 A resolution. PA1167 was found to form a glove-like beta-sandwich composed of 15 beta-strands and 3 alpha-helices. The structural difference between the beta-sandwich PA1167 of family PL-7 and alpha/alpha-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel beta-helix, alpha/alpha-barrel, and alpha/alpha-barrel + antiparallel beta-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide lyases.

Sphingomonas sp. A1 possesses a high molecular weight (HMW) alginate uptake system composed of a novel pit formed on the cell surface and a pit-dependent ATP-binding cassette (ABC) transporter in the inner membrane. The transportation of HMW alginate from the pit to the ABC transporter is mediated by the periplasmic HMW alginate-binding proteins AlgQ1 and AlgQ2. We determined the crystal structure of AlgQ2 complexed with an alginate tetrasaccharide using an alginate-free (apo) form as a search model and refined it at 1.6-A resolution. One tetrasaccharide was found between the N and C-terminal domains, which are connected by three extended hinge loops. The tetrasaccharide complex took on a closed domain form, in contrast to the open domain form of the apo form. The tetrasaccharide was bound in the cleft between the domains through van der Waals interactions and the formation of hydrogen bonds. Among the four sugar residues, the nonreducing end residue was located at the bottom of the cleft and exhibited the largest number of interactions with the surrounding amino acid residues, suggesting that AlgQ2 mainly recognizes and binds to the nonreducing part of a HMW alginate and delivers the polymer to the ABC transporter through conformational changes (open and closed forms) of the two domains.

Sphingomonas sp. A1 possesses a high molecular mass (average 25,700 Da) alginate uptake system mediated by a novel pit-dependent ABC transporter. The X-ray crystallographic structure of AlgQ2 (57,200 Da), an alginate-binding protein in the system, was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 18.3% for 15 to 2.0 A resolution data. The refined structure of AlgQ2 was comprised of 492 amino acid residues, 172 water molecules, and one calcium ion. AlgQ2 was composed of two globular domains with a deep cleft between them, which is expected to be the alginate-binding site. The overall structure is basically similar to that of maltose/maltodextrin-binding protein, except for the presence of an N2-subdomain. The entire calcium ion-binding site is similar to the site in the EF-hand motif, but comprises a ten residue loop. This calcium ion-binding site is about 40 A away from the alginate-binding site., (Copyright 2002 Elsevier Science Ltd.)

Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10Å resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif. [Copyright &y& Elsevier]