Crystal structures of Aspergillus oryzae Rib2 deaminase: the functional mechanism involved in riboflavin biosynthesis.

Riboflavin serves as the direct precursor of the FAD/FMN coenzymes and is biosynthesized in most prokaryotes, fungi and plants. Fungal Rib2 possesses a deaminase domain for deamination of pyrimidine in the third step of riboflavin biosynthesis. Here, four high-resolution crystal structures of a Rib2 deaminase from Aspergillus oryzae (AoRib2) are reported which display three distinct occluded, open and complex forms that are involved in substrate binding and catalysis. In addition to the deaminase domain, AoRib2 contains a unique C-terminal segment which is rich in charged residues. Deletion of this unique segment has no effect on either enzyme activity or protein stability. Nevertheless, the C-terminal αF helix preceding the segment plays a role in maintaining protein stability and activity. Unexpectedly, AoRib2 is the first mononucleotide deaminase found to exist as a monomer, perhaps due to the assistance of its unique longer loops (Lß1-ß2, LαB-ß3 and LαC-ß4). These results form the basis for a molecular understanding of riboflavin biosynthesis in fungi and might assist in the development of antibiotics.

Evolution of archaeal Rib7 and eubacterial RibG reductases in riboflavin biosynthesis: Substrate specificity and cofactor preference.

Archaeal/fungal Rib7 and eubacterial RibG possess a reductase domain for ribosyl reduction in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for an amino and a carbonyl group of the pyrimidine ring, respectively. Here, several crystal structures of Methanosarcina mazei Rib7 are reported at 2.27-1.95 Šresolution, which are the first archaeal dimeric Rib7 structures. Mutational analysis displayed that no detectable activity was observed for the Bacillus subtilis RibG K151A, K151D, and K151E mutants, and the M. mazei Rib7 D33N, D33K, and E156Q variants, while 0.1-0.6% of the activity was detected for the M. mazei Rib7 N9A, S29A, D33A, and D57N variants. Our results suggest that Lys151 in B. subtilis RibG, while Asp33 together with Arg36 in M. mazei Rib7, ensure the specific substrate recognition. Unexpectedly, an endogenous NADPH cofactor is observed in M. mazei Rib7, in which the 2'-phosphate group interacts with Ser88, and Arg91. Replacement of Ser88 with glutamate eliminates the endogenous NADPH binding and switches preference to NADH. The lower melting temperature of ∼10 °C for the S88E and R91A mutants suggests that nature had evolved a tightly bound NADPH to greatly enhance the structural stability of archaeal Rib7.

The N253F mutant structure of trehalose synthase from Deinococcus radiodurans reveals an open active-site topology.

Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures of Deinococcus radiodurans TS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes.

Structures of trehalose synthase from Deinococcus radiodurans reveal that a closed conformation is involved in catalysis of the intramolecular isomerization.

Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (ß/α)8 barrel, subdomain B, a C-terminal ß domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the -1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

Crystallization and preliminary crystallographic analysis of poly(3-hydroxybutyrate) depolymerase from Bacillus thuringiensis.

Poly[(R)-3-hydroxybutyrate] (PHB) is a microbial biopolymer that has been commercialized as biodegradable plastics. The key enzyme for the degradation is PHB depolymerase (PhaZ). A new intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) has been screened for potential applications in polymer biodegradation. Recombinant BtPhaZ was crystallized using 25% polyethylene glycol 3350, 0.2 M ammonium acetate, 0.1 M bis-tris pH 6.5 at 288 K. The crystals belonged to space group P1, with unit-cell parameters a = 42.97, b = 83.23, c = 85.50 Å, α = 73.45, ß = 82.83, γ = 83.49°. An X-ray diffraction data set was collected to 1.42 Šresolution with an Rmerge of 6.4%. Unexpectedly, a molecular-replacement solution was obtained using the crystal structure of Streptomyces lividans chloroperoxidase as a template, which shares 24% sequence identity to BtPhaZ. This is the first crystal structure of an intracellular poly(3-hydroxybutyrate) depolymerase.

Evolution of vitamin B2 biosynthesis: eubacterial RibG and fungal Rib2 deaminases.

Eubacterial RibG and yeast Rib2 possess a deaminase domain for pyrimidine deamination in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for ribose and ribitol, respectively. Here, the crystal structure of Bacillus subtilis RibG in complex with a deaminase product is reported at 2.56 Å resolution. Two loops move towards the product on substrate binding, resulting in interactions with the ribosyl and phosphate groups and significant conformational changes. The product carbonyl moiety is bent out of the pyrimidine ring to coordinate to the catalytic zinc ion. Such distortions in the bound substrate and product may play an essential role in enzyme catalysis. The yeast Rib2 structure was modelled and a mutational analysis was carried out in order to understand the mechanism of substrate recognition in these two enzymes. Detailed structural comparisons revealed that the two consecutive carbonyl backbones that occur prior to the PCXXC signature constitute a binding hole for the target amino group of the substrate. This amino-binding hole is essential in B. subtilis RibG and is also conserved in the RNA/DNA-editing deaminases.

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis.

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.

Various cross-reactivity of the grass pollen group 4 allergens: crystallographic study of the Bermuda grass isoallergen Cyn d 4.

The structure of Cyn d 4, the group 4 allergen from Bermuda grass, is reported at 2.15 Å resolution and is the first crystal structure of a naturally isolated pollen allergen. A conserved N-terminal segment that is only present in the large isoallergens forms extensive interactions with surrounding residues and hence greatly enhances the structural stability of the protein. Cyn d 4 contains an FAD cofactor that is covalently linked to His88 and Cys152. To date, all identified bicovalent flavoproteins are oxidases and their substrates are either sugars or secondary metabolites. A deep large hydrophobic substrate-binding cleft is present. Thus, Cyn d 4 may be an oxidase that is involved in the biosynthesis of a pollen-specific metabolite. Cyn d 4 shares ~70% sequence identity with the Pooideae group 4 allergens. Various cross-reactivities between grass pollen group 4 allergens have previously been demonstrated using sera from allergic patients. The protein surface displays an unusually large number of positively charged clusters, reflecting the high pI of ~10. 38 decapeptides that cover the solvent-accessible sequences did not show any significant IgE-binding activity using sera with high Cyn d 4 reactivity from four patients, suggesting that the IgE epitopes of Cyn d 4 are predominantly conformational in nature. Several group 4 structures were then modelled and their potential cross-reactive and species-specific IgE epitopes were proposed.

Structure-based discovery of triphenylmethane derivatives as inhibitors of hepatitis C virus helicase.

Hepatitis C virus nonstructural protein 3 (HCV NS3) helicase is believed to be essential for viral replication and has become an attractive target for the development of antiviral drugs. A fluorescence resonant energy transfer helicase assay was established for fast screening of putative inhibitors selected from virtual screening using the program DOCK. Soluble blue HT (1) was first identified as a novel HCV helicase inhibitor. Crystal structure of the NS3 helicase in complex with soluble blue HT shows that the inhibitor bears a significantly higher binding affinity mainly through a 4-sulfonatophenylaminophenyl group, and this is consistent with the activity assay. Subsequently, fragment-based searches were utilized to identify triphenylmethane derivatives for more potent inhibitors. Lead optimization resulted in a 3-bromo-4-hydroxyl substituted derivative 12 with an EC(50) value of 2.72 microM to Ava.5/Huh-7 cells and a lower cytotoxicity to parental Huh-7 cells (CC(50) = 10.5 microM), and it indeed suppressed HCV replication in the HCV replicon cells. Therefore, these inhibitors with structural novelty may serve as a useful scaffold for the discovery of new HCV NS3 helicase inhibitors.

Complex structure of Bacillus subtilis RibG: the reduction mechanism during riboflavin biosynthesis.

Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu51 and Lys79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-A resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu290, instead of the previously proposed Asp199, would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.

Functional roles of the 6-S-cysteinyl, 8alpha-N1-histidyl FAD in glucooligosaccharide oxidase from Acremonium strictum.

The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8alpha-positions of the flavin isoalloxazine ring cross-linked to Cys(130) and His(70), respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of approximately +69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with k(cat) values reduced to approximately 2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.

Crystal structure of a bifunctional deaminase and reductase from Bacillus subtilis involved in riboflavin biosynthesis.

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-A resolution displayed a tetrameric ring-like structure with an extensive interface of approximately 2400 A(2)/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.

Structural characterization of glucooligosaccharide oxidase from Acremonium strictum.

Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.

Directed evolution of Streptomyces clavuligerus deacetoxycephalosporin C synthase for enhancement of penicillin G expansion.

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the kcat/Km ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a kcat/Km ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.

Crystal structure of glucooligosaccharide oxidase from Acremonium strictum: a novel flavinylation of 6-S-cysteinyl, 8alpha-N1-histidyl FAD.

Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello- and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxy- cellobiono-1,5-lactam at 1.55- and 1.98-A resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8alpha-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C(6) atom and the 8alpha-methyl group of the isoalloxazine ring with Cys(130) and His(70), respectively. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. The complex structure suggests that this enzyme may prefer a beta-d-glucosyl residue at the reducing end with the conserved Tyr(429) acting as a general base to abstract the OH(1) proton in concert with the H(1) hydride transfer to the flavin N(5). Finally, a detailed comparison illustrates the structural conservation as well as the divergence between this protein and its related flavoenzymes.

The crystal structure of Ym1 at 1.31 A resolution.

Upon nematode infection, murine peritoneal macrophages synthesize and secrete large amounts of the Ym1 protein, which is a unique functional marker for alternatively activated macrophages in T(H)2-mediated inflammatory responses. Ym1 shares significant structural similarity to the family 18 chitinases. Previously, Ym1 has been studied with respect to its carbohydrate-binding ability and glycosyl hydrolysis activity and this has led to various inconclusive interpretations. Our present co-crystallization and soaking experiments with various glucosamine or N-acetylglucosamine oligomers yield only the uncomplexed Ym1. The refined Ym1 structure at 1.31A resolution clearly displays a water cluster forming an extensive hydrogen bond network with the "active-site" residues. This water cluster contributes notable electron density to lower resolution maps and this might have misled and given rise to a previous proposal for a monoglucosamine-binding site for Ym1. A structural comparison of family 18 glycosidase (-like) proteins reveals a lack of several conserved residues in Ym1, and illustrates the versatility of the divergent active sites. Therefore, Ym1 may lack N-acetylglucosamine-binding affinity, and this suggests that a new direction should be taken to unravel the function of Ym1.

Family shuffling of expandase genes to enhance substrate specificity for penicillin G.

Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

Crystal structure of Bacillus subtilis guanine deaminase: the first domain-swapped structure in the cytidine deaminase superfamily.

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.

Crystallization and preliminary crystallographic analysis of Bacillus subtilis guanine deaminase.

Guanine deaminase, a key enzyme in nucleotide metabolism, catalyzes the hydrolytic deamination of guanine to xanthine. The first guanine deaminase crystal from Bacillus subtilis was grown in the absence or presence of the inhibitor hypoxanthine in 30% polyethylene glycol 4000, 0.2 M ammonium acetate and 0.1 M sodium citrate pH 6.5. The crystals belong to space group C222(1), with unit-cell parameters a = 84.91, b = 90.90, c = 80.19 angstroms, with one dimer per asymmetric unit. The crystals diffract X-rays to beyond 1.2 angstroms resolution and an initial atomic model has been built based on selenomethionyl multiwavelength anomalous data at 2 angstroms resolution. Unexpectedly, this is the first domain-swapped structure in the cytidine deaminase superfamily.

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation.

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.