Bunzo Mikami

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.

Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snailAmpullaria crosseanwas expressed using aPichia pastorisexpression system and the crystal structure of the apo form was determined at 1.00 Å resolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double-ψ six-stranded β-barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high-resolution structures of EG27II provide a large amount of information for structure-based enzyme modification and cell-surface engineering, which will advance biofuel production using animal-derived cellulases.

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 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 import activity 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. 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. IMPORTANCE (118/120 words): The PTS identified in this work imports sulfate group-free unsaturated hyaluronan disaccharide as a result of the phosphorylation of the substrate at the C-6 position. S. agalactiae can be indigenous to animal hyaluronan-rich tissues owing to the bacterial molecular system for fragmentation, import, degradation, and metabolism of hyaluronan. Distinct from hyaluronan, most GAGs, which are sulfated at the C-6 position, are unsuitable for PTS due to its inability to phosphorylate the substrate. More recently, we have identified a solute-binding protein-dependent ABC transporter in a pathogenic Streptobacillus moniliformis as an importer of sulfated and non-sulfated fragmented GAGs without any substrate modification. Our findings regarding PTS and ABC transporter shed light on bacterial clever colonization/infection system targeting various animal GAGs.

Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy—key subunit interface redesign (KSIR)—that alters a natural subunit–subunit interface by selective deletion of a small number of “silent” amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

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.

-1,4-Mannanase (EC 3.2.1.78) catalyzes the hydrolysis of -1,4-glycosidic bonds within mannan, a major constituent group of the hemicelluloses. Bivalves and gastropods possess -1,4-mannanase and may degrade mannan in seaweed and/or phytoplankton to obtain carbon and energy using the secreted enzymes in their digestive systems. In the present study, the crystal structure of AkMan, a gastropod -1,4-mannanase prepared from the common sea hare Aplysia kurodai, was determined at 1.05 Å resolution. This is the first report of the three-dimensional structure of a gastropod -1,4-mannanase. The structure was compared with bivalve -1,4-mannanase and the roles of residues in the catalytic cleft were investigated. No obvious binding residue was found in subsite +1 and the substrate-binding site was exposed to the molecular surface, which may account for the enzymatic properties of mannanases that can digest complex substrates such as glucomannan and branched mannan.

The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy-L-erythro-hex-4-ene-pyranosyluronate-(mannuronate)(2)-mannuronic acid] were determined by X-ray crystallography at around 2.2 Å resolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Å from an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites -3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites -1 and +1. In particular, the O(η) atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, O(η) of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism.