Bacteria with a mouth: Discovery and new insights into cell surface structure and macromolecule transport.

A bacterium with a "mouth"-like pit structure isolated for the first time in the history of microbiology was a Gram-negative rod, containing glycosphingolipids in the cell envelope, and named Sphingomonas sp. strain A1. The pit was dynamic, with repetitive opening and closing during growth on alginate, and directly included alginate concentrated around the pit, particularly by flagellins, an alginate-binding protein localized on the cell surface. Alginate incorporated into the periplasm was subsequently transferred to the cytoplasm by cooperative interactions of periplasmic solute-binding proteins and an ATP-binding cassette transporter in the cytoplasmic membrane. The mechanisms of assembly, functions, and interactions between the above-mentioned molecules were clarified using structural biology. The pit was transplanted into other strains of sphingomonads, and the pitted recombinant cells were effectively applied to the production of bioethanol, bioremediation for dioxin removal, and other tasks. Studies of the function of the pit shed light on the biological significance of cell surface structures and macromolecule transport in bacteria.

The role of calcium binding to the EF-hand-like motif in bacterial solute-binding protein for alginate import.

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.

Polyphosphate-dependent nicotinamide adenine dinucleotide (NAD) kinase: A novel missing link in human mitochondria.

Polyphosphate [poly(P)] is described as a homopolymer of inorganic phosphates. Nicotinamide adenine dinucleotide kinase (NAD kinase) catalyzes the phosphorylation of NAD+ to NADP+ in the presence of ATP (ATP-NAD kinase). Novel NAD kinase that explicitly phosphorylates NAD+ to NADP+ using poly(P), besides ATP [ATP/poly(P)-NAD kinase], was found in bacteria, in particular, Gram-positive bacteria, and the gene encoding ATP/poly(P)-NAD kinase was also newly identified in Mycobacterium tuberculosis H37Rv. Both NAD kinases required multi-homopolymeric structures for activity expression. The enzymatic and genetic results, combined with their primary and tertiary structures, have led to the discovery of a long-awaited human mitochondrial NAD kinase. This discovery showed that the NAD kinase is a bacterial type of ATP/poly(P)-NAD kinase. These pioneering findings, i.e., ATP/poly(P)-NAD kinase, NAD kinase gene, and human mitochondrial NAD kinase, have significantly enhanced research on the biochemistry, molecular biology, and evolutionary biology of NAD kinase, mitochondria, and poly(P), including some biotechnological knowledge applicable to NADP+ production.

Hsp104-dependent ability to assimilate mannitol and sorbitol conferred by a truncated Cyc8 with a C-terminal polyglutamine in Saccharomyces cerevisiae.

Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeast Saccharomyces cerevisiae is unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation in TUP1 or CYC8. In this study, we found that spontaneous mutation of TUP1 or CYC8 also permitted assimilation of sorbitol. Some spontaneous nonsense mutations of CYC8 produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction of CYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations of TUP1 or CYC8 was guanidine hydrochloride-tolerant. It is physiologically reasonable that S. cerevisiae carries this mechanism to acquire the ability to assimilate major polyols in nature.

Uncovering the reactive nature of 4-deoxy-L-erythro-5-hexoseulose uronate for the utilization of alginate, a promising marine biopolymer.

Alginate is a linear polyuronate in brown macroalgae. It is also a promising marine biopolymer that can be degraded by exo-type alginate lyase into an unsaturated uronate that is non-enzymatically or enzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronate (DEH). In a bioengineered yeast Saccharomyces cerevisiae (DEH++) strain that utilizes DEH, DEH is not only an important physiological metabolite but also a promising carbon source for biorefinery systems. In this study, we uncovered the essential chemical nature of DEH. In particular, we showed that DEH non-enzymatically reacts with specific amino groups in Tris, ammonium salts [(NH4)2SO4 and NH4Cl], and certain amino acids (e.g., Gly, Ser, Gln, Thr, and Lys) at 30 °C and forms other compounds, one of which we tentatively named DEH-related product-1 (DRP-1). In contrast, Asn, Met, Glu, and Arg were almost inert and Ala, Pro, Leu, Ile, Phe, Val, and Asp, as well as sodium nitrate (NaNO3), were inert in the presence of DEH. Some of the above amino acids (Asn, Glu, Ala, Pro, Phe, and Asp) were suitable nitrogen sources for the DEH++ yeast strain, whereas ammonium salts and Ser, Gln, and Thr were poor nitrogen sources owing to their high reactivity to DEH. Nutrient-rich YP medium with 1% (w/v) Yeast extract and 2% (w/v) Tryptone, as well as 10-fold diluted YP medium, could also be effectively used as nitrogen sources. Finally, we identified DRP-1 as a 2-furancarboxylic acid and showed that it has a growth-inhibitory effect on the DEH++ yeast strain. These results show the reactive nature of DEH and suggest a basis for selecting nitrogen sources for use with DEH and alginate in biorefineries. Our results also provide insight into the physiological utilization of DEH. The environmental source of 2-furancarboxylic acid is also discussed.

Streptococcal phosphotransferase system imports unsaturated hyaluronan disaccharide derived from host extracellular matrices.

Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS activity for import of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides.

Substrate recognition by bacterial solute-binding protein is responsible for import of extracellular hyaluronan and chondroitin sulfate from the animal host.

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.

Structural studies on bacterial system used in the recognition and uptake of the macromolecule alginate.

Alginate is an acidic heteropolysaccharide produced by brown seaweed and certain kinds of bacteria. The cells of Sphingomonas sp. strain A1, a gram-negative bacterium, have several alginate-degrading enzymes in their cytoplasm and efficiently utilize this polymer for their growth. Sphingomonas sp. strain A1 cells can directly incorporate alginate into their cytoplasm through a transport system consisting of a "pit" on their cell surface, substrate-binding proteins in their periplasm, and an ATP-binding cassette transporter in their inner membrane. This review deals with the structural and functional aspects of bacterial systems necessary for the recognition and uptake of alginate.

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis.

The currently available haemoglobin A1c (HbA1c) enzymatic assay consists of two specific steps: proteolysis of HbA1c and oxidation of the liberated fructosyl peptide by fructosyl peptide oxidase (FPOX). To develop a more convenient and high throughput assay, we devised novel protease-free assay system employing modified FPOX with HbA1c oxidation activity, namely HbA1c direct oxidase (HbA1cOX). AnFPOX-15, a modified FPOX from Aspergillus nidulans, was selected for conversion to HbA1cOX. As deduced from the crystal structure of AnFPOX-15, R61 was expected to obstruct the entrance of bulky substrates. An R61G mutant was thus constructed to open the gate at the active site. The prepared mutant exhibited significant reactivity for fructosyl hexapeptide (F-6P, N-terminal amino acids of HbA1c), and its crystal structure revealed a wider gate observed for AnFPOX-15. To improve the reactivity for F-6P, several mutagenesis approaches were performed. The ultimately generated AnFPOX-47 exhibited the highest F-6P reactivity and possessed HbA1c oxidation activity. HbA1c levels in blood samples as measured using the direct assay system using AnFPOX-47 were highly correlated with the levels measured using the conventional HPLC method. In this study, FPOX was successfully converted to HbA1cOX, which could represent a novel in vitro diagnostic modality for diabetes mellitus.

Probiotics in human gut microbiota can degrade host glycosaminoglycans.

Glycosaminoglycans (GAGs) (e.g. heparin, chondroitin sulfate, and hyaluronan) show various significant physiological functions as a major component of extracellular matrix in animals. Some bacteria target GAGs for adhesion and/or infection to host cells, although no probiotics have been known to degrade GAGs. Here, we show GAG degradation by probiotics from human gut microbiota and their adhesion to human intestinal cells through a GAG. GAG-degrading bacteria were isolated from human faeces and identified as Enterococcus faecium, and some typical probiotics such as Lactobacillus casei, Lactobacillus rhamnosus and Enterococcus faecalis were also found to degrade heparin. GAG-degrading lactobacilli and enterococci including the isolated E. faecium possessed a genetic cluster encoding GAG-degrading/metabolising enzymes in the bacterial genome. KduI and KduD enzymes encoded in the GAG cluster of L. rhamnosus functioned as 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase and 2-keto-3-deoxy-d-gluconate dehydrogenase, respectively, both of which were crucial for GAG metabolism. GAG-degrading L. rhamnosus and E. faecium attached to human intestinal Caco-2 cells via heparin. Some species of Bacteroides, considered to be the next generation probiotics, degraded chondroitin sulfate C and hyaluronan, and genes coding for the Bacteroides GAG-degrading enzyme were frequently detected from human gut microbiota. This is the first report on GAG-degrading probiotics in human gut microbiota.

Alternative substrate-bound conformation of bacterial solute-binding protein involved in the import of mammalian host glycosaminoglycans.

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.

Binding mode of metal ions to the bacterial iron import protein EfeO.

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.

A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate.

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.

Crucial role of 4-deoxy-L-erythro-5-hexoseulose uronate reductase for alginate utilization revealed by adaptive evolution in engineered Saccharomyces cerevisiae.

In brown macroalgae, alginate and D-mannitol are promising carbohydrates for biorefinery. Saccharomyces cerevisiae is widely used as a microbial cell factory, but this budding yeast is unable to utilize either alginate or D-mannitol. Alginate can be depolymerized by both endo-type and exo-type alginate lyases, yielding a monouronate, 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a key intermediate in the metabolism of alginate. Here, we constructed engineered two S. cerevisiae strains that are able to utilize both DEH and D-mannitol on two different strain backgrounds, and we also improved their aerobic growth in a DEH liquid medium through adaptive evolution. In both evolved strains, one of the causal mutations was surprisingly identical, a c.50A > G mutation in the codon-optimized NAD(P)H-dependent DEH reductase gene, one of the 4 genes introduced to confer the capacity to utilize DEH. This mutation resulted in an E17G substitution at a loop structure near the coenzyme-binding site of this reductase, and enhanced the reductase activity and aerobic growth in both evolved strains. Thus, the crucial role for this reductase reaction in the metabolism of DEH in the engineered S. cerevisiae is demonstrated, and this finding provides significant information for synthetic construction of a S. cerevisiae strain as a platform for alginate utilization.

A bacterial ABC transporter enables import of mammalian host glycosaminoglycans.

Glycosaminoglycans (GAGs), such as hyaluronan, chondroitin sulfate, and heparin, constitute mammalian extracellular matrices. The uronate and amino sugar residues in hyaluronan and chondroitin sulfate are linked by 1,3-glycoside bond, while heparin contains 1,4-glycoside bond. Some bacteria target GAGs as means of establishing colonization and/or infection, and bacterial degradation mechanisms of GAGs have been well characterized. However, little is known about the bacterial import of GAGs. Here, we show a GAG import system, comprised of a solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter, in the pathogenic Streptobacillus moniliformis. A genetic cluster responsible for depolymerization, degradation, and metabolism of GAGs as well as the ABC transporter system was found in the S. moniliformis genome. This bacterium degraded hyaluronan and chondroitin sulfate with an expression of the genetic cluster, while heparin repressed the bacterial growth. The purified recombinant Smon0123 exhibited an affinity with disaccharides generated from hyaluronan and chondroitin sulfate. X-ray crystallography indicated binding mode of Smon0123 to GAG disaccharides. The purified recombinant ABC transporter as a tetramer (Smon0121-Smon0122/Smon0120-Smon0120) reconstructed in liposomes enhanced its ATPase activity in the presence of Smon0123 and GAG disaccharides. This is the first report that has molecularly depicted a bacterial import system of both sulfated and non-sulfated GAGs.

Extremely high intracellular concentration of glucose-6-phosphate and NAD(H) in Deinococcus radiodurans.

Deinococcus radiodurans is highly resistant to ionizing radiation and UV radiation, and oxidative stress caused by such radiations. NADP(H) seems to be important for this resistance (Slade and Radman, Microbiol Mol Biol Rev 75:133-191; Slade, Radman, Microbiol Mol Biol Rev 75:133-191, 2011), but the mechanism underlying the generation of NADP(H) or NAD(H) in D. radiodurans has not fully been addressed. Intracellular concentrations of NAD+, NADH, NADP+, and NADPH in D. radiodurans are also not determined yet. We found that cell extracts of D. radiodurans catalyzed reduction of NAD(P)+ in vitro, indicating that D. radiodurans cells contain both enzymes and a high concentration of substrates for this activity. The enzyme and the substrate were attributed to glucose-6-phosphate dehydrogenase and glucose-6-phosphate of which intracellular concentration was extremely high. Unexpectedly, the intracellular concentration of NAD(H) was also much greater than that of NADP(H), suggesting some significant roles of NADH. These unusual features of this bacterium would shed light on a new aspect of physiology of this bacterium.

Lateral-typed flagellin responsible for formation of a polar flagellum but not of lateral flagella in Sphingomonas sp. strain A1.

Alginate-assimilating Sphingomonas sp. strain A1 is the Gram-negative bacterium first identified to form a single polar flagellum containing lateral-typed flagellin (p6) in the filament. In addition to the p6 flagellin, two polar-typed flagellins (p5 and p5') are also included in the flagellum. Here we show the significant role of p6 as well as p5/p5' in flagellum formation and cell motility towards alginate. A p6 gene disruptant significantly reduced flagellum formation and it showed no cell motility, whereas each mutant with a disruption in the p5 or p5' gene exhibited cell motility through the formation of a polar flagellum containing p6. The ratio of p6 to p5 decreased in proportion to cell growth, suggesting that strain A1 changes flagellin ratios in the filament depending on the external environment. Each of purified recombinant p5 and p6 proteins formed the filament by in vitro self-assembly and an anti-p5 antibody reacted with the p5 filament but not with the p6 filament. Immunoelectron microscopy using an anti-p5 antibody indicated that strain A1 formed two types of the filament in a single polar flagellum: p6 alone in the entire filament and p5 elongation filament subsequent to the p6 proximal end. Immunoprecipitation with an anti-p5 antibody directly demonstrated that p5 and p6 coexist in a single filament. Strain A1 cells were also found to exhibit a chemotactic motility in response to alginate. This is the first report on function/location of the lateral-typed flagellin in a single polar flagellum and the bacterial chemotaxis towards alginate.

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

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.

Structural determinants in bacterial 2-keto-3-deoxy-D-gluconate dehydrogenase KduD for dual-coenzyme specificity.

Short-chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2-keto-3-deoxy-d-gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG-producing SDR enzymes (NADPH-dependent A1-R and NADH-dependent A1-R') involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes-KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)-prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5-diketo-3-deoxy-d-gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X-ray crystallography. Both PcaKduD and SpyDhuD consist of a three-layered structure, α/ß/α, with a coenzyme-binding site in the Rossmann fold; similar to enzymes A1-R and A1-R', both arrange the two short and long loops close to the coenzyme-binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1-R' but not A1-R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2' coenzyme region were determined to be structurally crucial for dual-coenzyme specificity in PcaKduD by structural comparison of the NADH- and NADPH-specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual-coenzyme specificity. Proteins 2016; 84:934-947. © 2016 Wiley Periodicals, Inc.

Biofuel Production Based on Carbohydrates from Both Brown and Red Macroalgae: Recent Developments in Key Biotechnologies.

Marine macroalgae (green, red and brown macroalgae) have attracted attention as an alternative source of renewable biomass for producing both fuels and chemicals due to their high content of suitable carbohydrates and to their advantages over terrestrial biomass. However, except for green macroalgae, which contain relatively easily-fermentable glucans as their major carbohydrates, practical utilization of red and brown macroalgae has been regarded as difficult due to the major carbohydrates (alginate and mannitol of brown macroalgae and 3,6-anhydro-L-galactose of red macroalgae) not being easily fermentable. Recently, several key biotechnologies using microbes have been developed enabling utilization of these brown and red macroalgal carbohydrates as carbon sources for the production of fuels (ethanol). In this review, we focus on these recent developments with emphasis on microbiological biotechnologies.