Arthrobacter globiformis I42 Glucodextranase (1UG9, 1ULV)
 A glucodextranase (iGDase) from Arthrobacter globiformis I42 hydrolyzes alpha-1,6-glucosidic linkages of dextran from the non-reducing end to produce beta-D-glucose via an inverting reaction mechanism and classified into the glycoside hydrolase family 15 (GH15). Here we determined the crystal structures of iGDase of the unliganded form and the complex with acarbose at 2.42-A resolution. The structure of iGDase is composed of four domains N, A, B, and C. Domain A forms an (alpha/alpha)(6)-barrel structure and domain N consists of 17 antiparallel beta-strands, and both domains are conserved in bacterial glucoamylases (GAs) and appear to be mainly concerned with catalytic activity. The structure of iGDase complexed with acarbose revealed that the positions and orientations of the residues at subsites -1 and +1 are nearly identical between iGDase and GA; however, the residues corresponding to subsite 3, which form the entrance of the substrate binding pocket, and the position of the open space and constriction of iGDase are different from those of GAs. On the other hand, domains B and C are not found in the bacterial GAs. The primary structure of domain C is homologous with a surface layer homology domain of pullulanases, and the three-dimensional structure of domain C resembles the carbohydrate-binding domain of some glycohydrolases.
J. Biol. Chem. (2004) 279, 10575-10583.

Aspergillus niger
Isoplullanase (1WMR)

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H(f) to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-A resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 beta-strands and forms a beta-sandwich. Domain C, where the active site is located, forms a right-handed beta-helix, and the lengths of the pitches of each coil of the beta-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites +2 and +3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the beta-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.
J. Mol. Biol. (2008) 376, 210-220.


Aspergillus oryzae
Aspartic proteinase (1IAD, 1IZE)

 The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9 Å resolution. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.
J. Mol. Biol. (2003) 326, 1503-1511.

Human and mouse Iba1 (2D58, 1WY9)

 Iba1 (ionized calcium-binding adaptor molecule 1) with 147 amino acid residues has been identified as a calcium (Ca2+)-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in Ca2+-free form and mouse Iba1 in Ca2+-bound form, to a resolution of 1.9 Å and 2.1 Å, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca2+, but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca2+-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, leading to the formation of a dimer in crystals, which demonstrates that Iba1 has a novel molecular switching mechanism dependent on Ca2+-binding, to interact with target molecules.
J. Mol. Biol. (2006) 364, 449-457.

Pseudomonas cichorii
Tagatose epimerase (2OU4)

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 A, respectively. A subunit of P. cichoriid-TE adopts a (beta/alpha)(8) barrel structure, and a metal ion (Mn(2+)) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the beta-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn(2+), and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.
J. Mol. Biol. (2007) 374, 443-453.

Pseudomonas stutzeri
Rhamnose Isomerase
(2HCV, 2I56, 2I57)

 The crystal structures of Pseudomonas stutzeri L-rhamnose isomerase (P. stutzeri L-RhI) and its complexes with L-rhamnose and D-allose have been determined at a resolution of 2.0 Å, 1.97 Å, and 1.97 Å, respectively. P. stutzeri L-RhI has a large domain with (b/a)8 barrel fold and an additional small domain composed of seven a-helices, forming a homo tetramer, as found in L-RhI from Escherichia coli and D-xylose isomerases (D-XIs) from various microorganisms. P. stutzeri L-RhI shows the broad substrate-specificity compared to that of E. coli L-RhI. The complex structures of the P. stutzeri L-RhI with L-rhamnose and D-allose show that both substrates are nicely fitted to the substrate-binding site of P. stutzeri L-RhI, and that the substrate-binding site structure of P. stutzeri L-RhI has similar partly to that of L-RhI and partly to that of D-XI.
J. Mol. Biol. (2007) 365, 1505-1516.

Sporobolomyces salmonicolor
Carbonyl Reductase (1ZZE, 1Y1P)

 The X-ray structures of red yeast Sporobolomyces salmonicolor carbonyl reductase (SSCR) and its complex with a coenzyme, NADPH, have been determined at a resolution of 1.8 Å and 1.6 Å, respectively. SSCR has two domains, an NADPH-binding domain and a substrate-binding domain, and belongs to the short-chain dehydrogenases/reductases family. The structure of the NADPH-binding domain and the interaction between the enzyme and NADPH are very similar to those found in other structure-solved enzymes belonging to the short-chain dehydrogenases/reductases family, while the structure of the substrate-binding domain is unique. SSCR has stereoselectivity in its catalytic reaction, giving rise to excessive production of (S)-alcohols from ethyl 4-chloro-3-oxobutanoate. The X-ray structure of the SSCR/NADPH complex and preliminary modeling show that the formation of the hydrophobic channel induced by the binding of NADPH is closely related to the stereoselective reduction by SSCR.
J. Mol. Biol. (2005) 352, 551-558.


Sulfolobus tokodaii Cytochrome P450 (1UEB)

 A cytochrome P450 from acidothermophilic archaebacterium Sulfolobus tokodaii strain7. S. tokodaii strain7 (P450st) carrying histidine6-tag has been expressed in E. coli and purified with high yield and homogeneity. The X-ray structure of P450st was determined at 3.0 Å resolution. Structural comparison with cytochrome P450 from Sulfolobus solfataricus (CYP119) suggests that the region from the F to G helices and the binding Cl- is possibly responsible to the affinity of a ligand coordinating to the heme iron. The direct electrochemistry of P450st in a DDAB film on the PFC electrode has been also demonstrated. The quasi-reversible redox response has been observed even at 80 °C.
J. Inorg. Biochem. (2004) 98, 1194-1199
.

Sulfolobus tokodaii
Molybdenum cofactor biosynthesis protein C, ST0472 (2OHD)

The crystal structure of a putative molybdenum-cofactor (Moco) biosynthesis protein C (MoaC) from Sulfolobus tokodaii (ST0472) was determined at 2.2 A resolution. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 123.31, b = 78.58, c = 112.67 A, beta = 118.1 degrees . The structure was solved by molecular replacement using the structure of Escherichia coli MoaC as the probe model. The asymmetric unit is composed of a hexamer arranged as a trimer of dimers with noncrystallographic 32 symmetry. The structure of ST0472 is very similar to that of E. coli MoaC; however, in the ST0472 protein an additional loop formed by the insertion of seven residues participates in intermonomer interactions and the new structure also reveals the formation of an interdimer beta-sheet. These features may contribute to the stability of the oligomeric state.
Acta Crystallogr. Sect F. (2008) 64, 589-592
.

Sulfolobus tokodaii Selenium-binding protein, ST0059 (2ECE)

A 56-kDa selenium-binding protein (SBP56) was originally discovered as a cytosolic protein with the selenium-binding activity (Bansal et al., 1989). SBP56 is a highly conserved protein found widely in
microorganisms, plants and animals. SBP56 was reported to be involved in the transport of selenium compounds and intra-Golgi transport, however, its function in microorganisms has been still unclear. To obtain new insights into structure-function relationship of SBP56, we have determined the X-ray structure of Sulfolobus tokodaii hypothetical SBP56 (ST0059), which has 39 % identity in amino acid sequence with human SBP56.


Thermoactinomyces vulgaris
R-47 alpha-Amylase 1 (1JT1 etc.
)
 The X-ray structures of complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) with an inhibitor acarbose and an inactive mutant TVAI with malto-hexaose and malto-tridecaose have been determined at 2.6, 2.0 and 1.8 Å resolutions. Acarbose binds to the catalytic site of TVAI, and interactions between acarbose and the enzyme are very similar to those found in other structure-solved a-amylase/acarbose complexes, supporting the proposed catalytic mechanism. Based on the structure of TVAI/acarbose complex, the binding mode of pullulan containing alpha-(1,6) glucoside linkages could be deduced. Besides the catalytic site, four sugar binding sites on the molecular surface are found in these X-ray structures. Two sugar binding sites in domain N hold the oligosaccharides with a regular helical structure of amylose, which suggests the domain N is a starch binding domain acting as an anchor to starch in the catalytic reaction of the enzyme. An assay of hydrolyzing activity for the raw starches confirmed that TVAI can efficiently hydrolyze raw starch.
J. Mol. Biol. (2004) 335, 811-822.

Thermoactinomyces vulgaris
R-47 alpha-Amylase 2 (1JT2 etc.
)
 Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) has the unique ability to hydrolyze cyclodextrins (CDs) with various sized cavities, as well as starch. To understand the relationship between structure and substrate specificity, X-ray structures of a TVAII/acarbose complex and inactive mutant TVAII (D325N·D421N)/alpha-, beta- and gamma-CDs complexes were determined at resolutions of 2.9 Å, 2.9 Å, 2.8 Å and 3.1 Å, respectively. In all complexes, the interactions between ligands and enzymes at subsites -1, -2 and -3 were almost the same, but striking differences in the catalytic site structure were found at subsites +1 and +2, where Trp356 and Tyr374 changed the conformation of the side chain depending on the structure and size of the ligands. Trp356 and Tyr374 are thought to be responsible for the multiple substrate-recognition mechanism of TVAII, providing the unique substrate specificity. In the beta-CD complex, the beta-CD maintains a regular conical structure, making it difficult for Glu354 to protonate the O4 atom at the hydrolyzing site as a previously proposed hydrolyzing mechanism of a-amylase. From the X-ray structures, it is suggested that the protonation of the O4 atom is possibly carried out via a hydrogen atom of the inter-glucose hydrogen bond at the hydrolyzing site.
J. Biol. Chem. (2004) 279, 31033-31040.

Thermoactinomyces vulgaris
R-47 Cyclodextrin binding protein (2DFZ)

 The crystal structure of Thermoactinomyces vulgaris cyclodextrin-binding protein (TvuCBP) complexed with gamma-cyclodextrin has been determined. Like E. coli maltodextrin-binding protein (EcoMBP) and other bacterial sugar-binding proteins, TvuCBP consists of two domains, N-domain and C-domain, both of which are composed of a central beta-sheet surrounded by alpha-helices, and the domains are joined by a hinge region containing three segments. Gmma-Cyclodextrin is located at a cleft formed by the two domains. A common functional conformational change has been reported in this protein family, which involves switching from an open form to a sugar-transporter bindable form, designated a closed form. The TvuCBP―gamma-cyclodextrin complex structurally resembles the closed form of EcoMBP, indicating that TvuCBP complexed with gamma-cyclodextrin adopts the closed form. Despite having similar folds, the sugar-binding site of N-domain of TvuCBP and other bacterial sugar-binding proteins are strikingly different. In TvuCBP, the side-chain of Leu59 protrudes into the sugar binding cleft and orients toward the central cavity of gamma-CD, thus Leu59 appears to play the key role in binding.
FEBS J. (2007) 274, 2109-2120.