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Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.

SummaryThe CRISPR-Cas system is the RNA-guided immune defense mechanism in bacteria and archaea. Csm1 belongs to the Cas10 family, which is the common signature protein of the type III system. Csm1 is the largest subunit of the Csm interference complex in the type III-A subtype, which targets foreign DNA or RNA. Here, we report crystallographic and biochemical analyses of Thermococcus onnurineus Csm1, revealing a five-domain organization and single-stranded DNA (ssDNA)-specific nuclease activity associated with the N-terminal HD domain. This domain folds into permuted secondary structures in comparison with the HD domain of Cas3 and contains all the catalytically important residues. It exhibited both endo- and exonuclease activities in an Ni2+ or Mn2+-dependent manner. The narrow width of the active-site cleft appears to restrict the substrate specificity to ssDNA and thus to prevent Csm1 from cleaving double-stranded chromosomal DNA. These data suggest that Csm1 may function in DNA interference by the Csm effector complex.

In meiotic DNA recombination, the Hop2−Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. Here, the crystal structure of Hop2−Mnd1 reveals that it forms a curved rod-like structure consisting of three leucine zippers and two kinked junctions. One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure. Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament. Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations. These findings allow us to propose a model explaining how Hop2−Mnd1 juxtaposes Dmc1-bound ssDNA with distorted recipient double-stranded DNA and thus facilitates strand invasion.

Eukaryotic structural maintenance of chromosomes (SMC)-kleisin complexes form large, ring-shaped assemblies that promote accurate chromosome segregation. Their asymmetric structural core comprises SMC heterodimers that associate with both ends of a kleisin subunit. However, prokaryotic condensin Smc-ScpAB is composed of symmetric Smc homodimers associated with the kleisin ScpA in a postulated symmetrical manner. Here, we demonstrate that Smc molecules have two distinct binding sites for ScpA. The N terminus of ScpA binds the Smc coiled coil, whereas the C terminus binds the Smc ATPase domain. We show that in Bacillus subtilis cells, an Smc dimer is bridged by a single ScpAB to generate asymmetric tripartite rings analogous to eukaryotic SMC complexes. We define a molecular mechanism that ensures asymmetric assembly, and we conclude that the basic architecture of SMC-kleisin rings evolved before the emergence of eukaryotes.

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1121334151, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C-terminal domain of PF1127 has a unique β-hairpin structure that protrudes out of an α-helix and contains several positively charged residues. We demonstrate that PF1127 binds double-stranded DNA and RNA and that this activity requires an intact β-hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β-hairpin structure and exhibits no DNA-binding properties under the same experimental conditions. Notably, the C-terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N-terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid-binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Proteins 2013. © 2012 Wiley Periodicals, Inc.

The surface of proteins can be charged with zinc ions and the anomalous signals from these zinc ions can be used for structure determination of proteins., Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination.

Many prokaryotic organisms acquire immunity against foreign genetic material by incorporating a short segment of foreign DNA called spacer into chromosomal loci, termed clustered regularly interspaced short palindromic repeats (CRISPRs). The encoded RNAs are processed into small fragments that guide the silencing of the invading genetic elements. The CRISPR-associated (Cas) proteins are the main executioners of these processes. Herein, we report the crystal structure of Stu0660 of Streptococcus thermophilus, a Cas protein involved in the acquisition of new spacers. By homotetramerization, Stu0660 forms a central channel which is decorated with basic amino acids and binds linear double-stranded DNA (dsDNA), but not circular dsDNA. Despite undetectably low sequence similarity, two N-terminal domains of Stu0660 are similar to the entire structure of an Enterococcus faecalis Csn2 protein, which also forms a homotetramer and binds dsDNA. Thus, this work identifies a previously unknown group of Stu0660-like Csn2 proteins (∼350 residues), which are larger than the known canonical Csn2 proteins (∼220 residues) by containing an extra C-terminal domain. The commonly present central channel in the two subgroups appears as a design to selectively interact with linear dsDNA.

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

The structural maintenance of chromosomes (SMC) family proteins are commonly found in the multiprotein complexes involved in chromosome organization, including chromosome condensation and sister chromatid cohesion. These proteins are characterized by forming a V-shaped homo- or heterodimeric structure with two long coiled-coil arms having two ATPase head domains at the distal ends. The hinge domain, located in the middle of the coiled coil, forms the dimer interface. In addition to being the dimerization module, SMC hinges appear to play other roles, including the gateway function for DNA entry into the cohesin complex. Herein, we report the homodimeric structure of the hinge domain of Escherichia coli MukB, which forms a prokaryotic condensin complex with two non-SMC subunits, MukE and MukF. In contrast with SMC hinge of Thermotoga maritima which has a sizable central hole at the dimer interface, MukB hinge forms a constricted dimer interface lacking a hole. Under our assay conditions, MukB hinge does not interact with DNA in accordance with the absence of a notable positively charged surface patch. The function of MukB hinge appears to be limited to dimerization of two copies of MukB molecules. Proteins 2010. © 2009 Wiley-Liss, Inc.

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k(cat)/K(m) value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor . Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.

Summary SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome., Graphical Abstract, Highlights • Prokaryotic Smc-ScpAB complexes form rod-like structures • Binding of ATP and DNA induces a rod-to-ring transition in prokaryotic condensin • The condensin hinge is rigidly anchored to its coiled coil • The rod-like conformation is a conserved feature of SMC protein dimers, Soh et al. show that the rod-like conformation is a conserved architectural scheme of SMC complexes. Upon ATP-induced binding to DNA, the juxtaposed coiled coils of prokaryotic Smc-ScpAB adopt an open conformation to expose a DNA binding site at the inner surface of the hinge domain.

Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article ( A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid , J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E 14 ) of vBcl-2 is critical for KSHV lytic replication. Mutating E 14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.

Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Angstrom resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k(cat) value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k(cat) value of R158K was similar to that of the wild type with the K(m) value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.

A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent-exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in Δ5-3-ketosteroid isomerase from Pseudomonas putida biotype B (KSI-PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side-chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI-PI, mutants of those amino acids associated with the SEHC were prepared. The W92A, L125A/V127A, and W92A/L125A/V127A mutations largely decreased the conformational stability, while the L125F/V127F mutation slightly increased the stability, indicating that hydrophobic packing by the SEHC is important in maintaining stability. The crystal structure of W92A revealed that the decreased stability caused by the removal of the bulky side-chain of Trp92 could be attributed to the destabilization of the surface hydrophobic layer consisting of a solvent-exposed β-sheet. Consistent with the structural data, the binding affinities for three different steroids showed that the surface hydrophobic layer stabilized by SEHC is required for KSI-PI to efficiently recognize hydrophobic steroids. Unfolding kinetics based on analysis of the ΦU value also indicated that the SEHC in the native state was resistant to the unfolding process, despite its solvent-exposed site. Taken together, our results demonstrate that the SEHC plays a key role in the structural integrity that is needed for KSI-PI to stabilize the hydrophobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic steroids in water solution.

The family of peptidoglycan recognition proteins (PGRPs) are associated with the recognition of the peptidoglycan of microbes and subsequent activation of signaling pathways for immune response. Here the crystal structure of Drosophila PGRP-LB is determined at a resolution of 2.0 A and shows an active-site cleft with a zinc cage. Poor conservation of surface residues at the cleft predicts a widely varying individual specificity of PGRPs for molecular patterns on microbial cell walls. At the back of this cleft is a putatively conserved distinctive groove. The location and mainly hydrophobic nature of the groove indicate that the back face serves for subsequent signaling after clustering of PGRP molecules by binding to polymeric cell wall components.

RbsD is the only protein whose biochemical function is unknown among the six gene products of the rbs operon involved in the active transport of ribose. FucU, a paralogue of RbsD conserved from bacteria to human, is also the only protein whose function is unknown among the seven gene products of the l-fucose regulon. Here we report the crystal structures of Bacillus subtilis RbsD, which reveals a novel decameric toroidal assembly of the protein. Nuclear magnetic resonance and other studies on RbsD reveal that the intersubunit cleft of the protein binds specific forms of d-ribose, but it does not have an enzyme activity toward the sugar. Likewise, FucU binds l-fucose but lacks an enzyme activity toward this sugar. We conclude that RbsD and FucU are cytoplasmic sugar-binding proteins, a novel class of proteins whose functional role may lie in helping influx of the sugar substrates.

A novel inhibitor against maltose-producing alpha-amylase was prepared via stepwise degradation of a high-molecular-weight acarbose (HMWA) using Thermus maltogenic amylase (ThMA). The structure of the purified inhibitor was determined to be alpha-D-glucopyranosyl-alpha-acarviosinyl-D-glucopyranose (GlcAcvGlc) by (13)C NMR and MALDI-TOF/MS. Progress curves of PNPG2 hydrolysis by various amylolytic enzymes, including MGase, ThMA, and CDase I-5, in the presence of acarbose or GlcAcvGlc indicated a slow-binding mode of inhibition. Analytical ultracentrifugation and X-ray crystallography analyses revealed that the presence of GlcAcvGlc increased the dimerization of ThMA. The formation of dimer complexed with GlcAcvGlc might induce a conformational change in ThMA, leading to a two-step inhibition process. The inhibition potency of GlcAcvGlc for MGase, ThMA, and CDase I-5 was 3 orders of magnitude higher than that of acarbose.

Both d - and l -isomers of N -(hydroxyaminocarbonyl)phenylalanine ( 1 ) were shown to have strong binding affinity towards carboxypeptidase A (CPA) with d - 1 being more potent than its enantiomer by 3-fold (Chung, S. J.; Kim, D. H. Bioorg. Med. Chem. 2001 , 9 , 185.). In order to understand the reversed stereochemical preference shown in the CPA inhibition, we have solved the crystal structures of CPA complexed with each enantiometer of 1 up to 1.75 A resolution. Inhibitor l - 1 whose stereochemistry belongs to the stereochemical series of substrate binds CPA like substrate does with its carbonyl oxygen coordinating to the active site zinc ion. Its hydroxyl is engaged in hydrogen bonding with the carboxylate of Glu-270. On the other hand, in binding of d - 1 to CPA, its terminal hydroxyl group is involved in interactions with the active site zinc ion and the carboxylate of Glu-270. In both CPA· 1 complexes, the phenyl ring in 1 is fitted in the substrate recognition pocket at the S 1 ′ subsite, and the carboxylate of the inhibitors forms bifurcated hydrogen bonds with the guanidinium moiety of Arg-145 and a hydrogen bond with the guanidinium of Arg-127. In the complex of CPA· d - 1 , the carboxylate of the inhibitor is engaged in hydrogen bonding with the phenolic hydroxyl of the down-positioned Tyr-248. While the l - 1 binding induces a concerted movement of the backbone amino acid residues at the active site, only the downward movement of Tyr-248 was noted when d - 1 binds to CPA.

Summary Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase “head” and a “hinge” dimerization domain connected by a 49 nm coiled-coil “arm.” The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms., Graphical Abstract, Highlights • Crystallography and in vivo cross-linking reveal the architecture of prokaryotic Smc • Juxtaposition of the Smc arms misaligns the two Smc ATPase domains • Smc head engagement mechanically opens an interarm space • A model for DNA loop extrusion driven by the SMC ATPase cycle is presented, By combining high-throughput in vivo cysteine cross-linking and crystallography, Diebold-Durand et al. construct a high-resolution model of full-length prokaryotic Smc. It reveals that the rod-shaped Smc dimer lacks chambers for DNA and features misaligned head domains. Smc head engagement mechanically opens an interarm space.

Background: Phytases hydrolyze phytic acid ( myo -inositol-hexakisphosphate) to less-phosphorylated myo -inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the β propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. Results: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. Conclusions: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo -inositol triphosphates from phytate and the hydrolysis of myo -inositol monophosphates.

Simmondsin, a material related to food intake inhibition from jojoba (Simmondsia chinensis), was transglycosylated by Bacillus stearothermophilus maltogenic amylase (BSMA) reaction with acarbose to synthesize an antiobese compound with hypoglycemic activity. Ten percent each of acarbose and simmondsin were mixed and incubated with BSMA at 55°C. Glycosylation products of simmondsin were observed by thin layer chromatography (TLC) and high performance ion chromatography (HPIC). The major transfer product was purified by using Biogel P-2 column. The structure was determined by matrix-assisted laser desorption ionization with time of flight (MALDI-TOF):mass spectrometry (MS) and 13 C-NMR. The major transglycosylation product was pseudotrisaccharide (PTS)-simmondsin, in which PTS was attached by an a-(1i6) glycosidic linkage to simmondsin. The administration of transglycosylated simmondsin with acarbose (200 mg:kg per day for 6 days) significantly reduced the food intake by 74%, comparable to 62% of simmondsin versus control in ob:ob mice. The transfer product (10 mg:kg) significantly suppressed the postprandial blood glucose response to starch (2 g:kg) by 68%, comparable to 60% of acarbose in Zucker fa:fa rats. The results indicated that the transfer products would be effective agents in lowering both food intake and blood glucose. © 2000 Elsevier Science B.V. All rights reserved.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 A crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as ∼30°C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.

Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no β-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A “descending molecular see-saw” model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testos...

A multi-subunit ribonucleoprotein complex termed the Cmr RNA-silencing complex recognizes and destroys viral RNA in the CRISPR-mediated immune defence mechanism in many prokaryotes using an as yet unclear mechanism. InArchaeoglobus fulgidus, this complex consists of six subunits, Cmr1–Cmr6. Here, the crystal structure of Cmr1 fromA. fulgidusis reported, revealing that the protein is composed of two tightly associated ferredoxin-like domains. The domain located at the N-terminus is structurally most similar to the N-terminal ferredoxin-like domain of the CRISPR RNA-processing enzyme Cas6 fromPyrococcus furiosus. An ensuing mutational analysis identified a highly conserved basic surface patch that binds single-stranded nucleic acids specifically, including the mature CRISPR RNA, but in a sequence-independent manner. In addition, this subunit was found to cleave single-stranded RNA. Together, these studies elucidate the structure and the catalytic activity of the Cmr1 subunit.

The heterodimeric Rag GTPases consisting of RagA (or RagB) and RagC (or RagD) are the key regulator activating the target of rapamycin complex 1 (TORC1) in response to the level of amino acids. The heterodimer between GTP-loaded RagA/B and GDP-loaded RagC/D is the most active form that binds Raptor and leads to the activation of TORC1. Here, we present the crystal structure of Gtr1p(GTP)-Gtr2p(GDP), the active yeast Rag GTPase heterodimer. The structure reveals that GTP-to-GDP conversion on Gtr2p results in a large conformational transition of this subunit, including a large scale rearrangement of a long segment whose corresponding region in RagA is involved in binding to Raptor. In addition, the two GTPase domains of the heterodimer are brought to contact with each other, but without causing any conformational change of the Gtr1p subunit. These features explain how the nucleotide-bound statuses of the two GTPases subunits switch the Raptor binding affinity on and off.

The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with [nFe-mS] clusters, n = 2, 3, 4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near v approximately 13 and 8 MHz that can be assigned, respectively, to "distant ENDOR" from 13C in natural-abundance (1.1%) and from 14N (the delta m1 = +/- 2 transitions); the nuclei involved in this phenomenon are remote from and have negligible hyperfine couplings to the cluster. In addition, a number of proteins show local 13C ENDOR signals with resolved hyperfine interactions; these are assigned to the beta carbons of cysteines bound to the cluster [A(13C) approximately 1.0 MHz]. Five proteins show resolved, local delta m1 = +/- 2 ENDOR signals from 14N with an isotropic hyperfine coupling, 0.4 less than or equal to A(14N) less than or equal to 1.0, similar to those seen in ESEEM studies; these most likely are associated with N-H...S hydrogen bonds to the cluster. Anabaena ferredoxin further shows a signal corresponding to A(14N) approximately 4 MHz. Quadrupole coupling constants are derived for both local and distant 14N signals. The interpretation of the data is supported by studies on 15N- and 13C-enriched ferredoxin (Fd) from Anabaena 7120, where the 15N signals can be clearly correlated with the corresponding 14N signals and where the 13C signals are strongly enhanced. Thus, the observation of 14N delta m1 = +/- 2 signals at Q-band provides a new technique for examining weak interactions with a cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to L-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward L-fucose but also for the pyranase activity toward D-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.

Drosophila rely entirely on an innate immune response to combat microbial infection. Diaminopimelic acid-containing peptidoglycan, produced by Gram-negative bacteria, is recognized by two receptors, PGRP-LC and PGRP-LE, and activates a homolog of transcription factor NF-kappaB through the Imd signaling pathway. Here we show that full-length PGRP-LE acted as an intracellular receptor for monomeric peptidoglycan, whereas a version of PGRP-LE containing only the PGRP domain functioned extracellularly, like the mammalian CD14 molecule, to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface. Interaction with the imd signaling protein was not required for PGRP-LC signaling. Instead, PGRP-LC and PGRP-LE signaled through a receptor-interacting protein homotypic interaction motif-like motif. These data demonstrate that like mammals, drosophila use both extracellular and intracellular receptors, which have conserved signaling mechanisms, for innate immune recognition.

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of Gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the DeltaG(U)(H(2)O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the k(cat)/K(M) value by about 8- to 96-fold, but also increased the K(D) value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 A resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.

Malonyl-CoA decarboxylase (MCD), which catalyzes the conversion of malonyl-CoA to acetyl-CoA, is an evolutionarily distinct and highly conserved enzyme. MCD does not share sequence homology with other known decarboxylases, while the enzymes from different species exhibit at least >30% sequence identity to each other. In order to provide a canonical structure of the enzyme for detailed study of its structure-function relationship, the MCD of Rhizobium leguminosarum bv. trifolii was overexpressed and crystallized. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 133.45, b = 127.10, c = 66.37 A. The asymmetric unit is likely to contain two molecules of MCD (molecular weight of 51 418 Da), with a crystal Volume per protein weight (V(M)) of 2.69 A(3) Da(-1) and a solvent content of about 54.3% by Volume. A native data set to 3.0 A resolution was obtained using a rotating-anode X-ray generator.

The three-dimensional structures of Δ5-3-ketosteroid isomerases from two different bacterial species have been determined. The structures reveal an unusually apolar active site, in which each of several competitive inhibitors of the enzyme are held by two hydrogen bonds with the general acids Tyr14 and Asp99, and by hydrophobic interactions. The hydrogen bond between the Tyr14 hydroxyl and the C3 oxyanion of a transition-state analog is a low-barrier hydrogen bond, as indicated by a highly deshielded nuclear magnetic resonance. Structural and other biochemical studies have enabled the proposal of a detailed catalytic mechanism for Δ5-3-ketosteroid isomerase and provided a major thrust towards understanding the mechanism not only in chemical terms but also in energetics terms.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.

The Escherichia coli high-affinity ribose-transport system consists of six proteins encoded by the rbs operon (rbsD, rbsA, rbsC, rbsB, rbsK and rbsR). Of the six components, RbsD is the only one whose function is unknown. In order to gain insights into the function of RbsD by structural analysis, we overexpressed and crystallized the protein as a first step toward this goal. RbsD was overexpressed in E. coli and crystallized using the hanging-drop vapour-diffusion method at 296 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 285.9, b = 92.3, c = 93.3 A, beta = 105.0 degrees. The unit cell is likely to contain 64 molecules of RbsD, with a crystal volume per protein mass (V(M)) of 2.43 A(3) Da(-1) and a solvent content of about 49.3% by volume. An equilibrium centrifugation analysis demonstrated that RbsD (MW = 15 292 Da) exists as an octamer in solution, suggesting that the asymmetric unit contains two octameric assemblies of RbsD. A native data set to 2.7 A resolution was obtained from a flash-cooled crystal.

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.

The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B have been characterized in their roles in steroid binding and catalysis. Kinetic and equilibrium binding analyses were carried out for the mutant enzymes with the substitutions Phe-54 --Ala or Leu, Phe-82 --Ala or Leu, and Trp-116 --Ala, Phe, or Tyr. The removal of their bulky, aromatic side chains at any of these three positions results in reduced k(cat), particularly when Phe-82 or Trp-116 is replaced by Ala. The results are consistent with the binding interactions of the aromatic residues with the bound steroid contributing to catalysis. All the mutations except the F82A mutation increase K(m); the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility that the phenyl ring at position 82 might be unfavorable for substrate binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a phenyl ring at position 82, and a nonpolar aromatic or small side chain at position 116 might be favorable for binding the reaction intermediate. In contrast to the increased K(D) for equilenin, the enzymes with any substitutions at positions 54 and 116 display a decreased K(D) for 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116 might be unfavorable for product binding. The crystal structure of F82A determined to 2.1-A resolution reveals that Phe-82 is important for maintaining the active site geometry. Taken together, our results demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to the stabilization of steroid species including substrate, intermediate, and product.

Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

A novel bacterial phytase from a Bacillus amyloliquefaciens strain was crystallized using the hanging-drop vapour-diffusion method. The amino-acid sequence of the enzyme does not show any homology to those of other known phytases or phosphatases, with the exception of a phytase from Bacillus subtilis. The enzyme exhibits a thermal stability which is strongly dependent on calcium ions. High-quality single crystals of the enzyme in the absence of calcium ions were obtained using a precipitant solution containing 20% 2-methyl-2, 4-pentanediol and 0.1 M MES (pH 6.5). Native diffraction data to 2.0 A resolution were obtained from a flash-frozen crystal at 110 K using a rotating-anode X-ray source. The crystals belong to space group P212121 with unit-cell dimensions a = 50.4, b = 64.1, c = 104. 2 A and contain one monomer per asymmetric unit. Structure determination using heavy-atom derivative crystals is in progress, along with an effort to crystallize the calcium ion bound form of the enzyme.

A novel maltogenic amylase from Bacillus stearothermophilus ET1, which has a dual activity of α-1,4- and α-1,6-glycosidic bond cleavages and α-1,6-glycosidic bond formation, was crystallized by using the hanging-drop vapor-diffusion method. The best crystals were obtained by employing a high concentration of protein (56 mg ml−1) and a precipitant containing 22% glycerol, 1.6 M ammonium sulfate in 0.1 M Tris–HCl (pH 8.5). Native diffraction data to 2.66 Å resolution have been obtained from crystals flash-frozen at 110 K. The crystals belong to the space group P212121 with unit-cell dimensions of a = 77.62, b = 121.23, c = 244.29 Å, and contain three or four protomers per asymmetric unit. Structure determination by multiple isomorphous replacement is in progress.

A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69,627-Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47-70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) 1-5 from an alkalophilic Bacillus 1-5 strain. In addition to starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an alpha-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The beta-cyclodextrin-hydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site-directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.

The delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 36.48 angstrom, b = 74.30 angstrom, c = 96.02 angstrom, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Angstrom resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.