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Bacteria depend on the ferrous iron transport (Feo) system for the uptake of ferrous iron (Fe

AST-3424/OBI-3424 (denoted by 3424) is a novel prodrug bis-alkylating agent activated by AKR1C3. AKR1C3 is overexpressed in many types of cancer, particularly in liver, non-small cell lung, gastric, renal and CRPC cancer. Currently 3424 is being studied in phase 1/2 clinical trials for the treatment of solid and hematologic cancers, and it represents potentially a novel, selective anti-cancer agent for multiple indications. In this study, AKR1C3-dependent activation of 3424 was investigated in vitro using recombinant human AKR1C3. AKR1C3-dependent cytotoxicity of 3424 was determined in a wide range of human cancer cell lines with different AKR1C3 expression levels. In addition, anti-tumor activity of 3424 was also investigated in a broad panel of CDX and PDX models. AKR1C3-dependent activation of prodrug 3424 was evident by monitoring the decrease of 3424 and generation of the active form, 2660. Kinetic analysis indicated that AKR1C3 exhibited higher catalytic efficiency towards 3424 compared to the physiological substrates. There was a strong correlation between 3424 cytotoxic potency and AKR1C3 expression. The racemic mixture induced DNA cross-linking in a concentration dependent manner. Tumor growth inhibition of 3424 was shown to be better than or comparable to the standard of care chemotherapy at clinically achievable doses as a single agent in various CDX models with high expression of AKR1C3, including liver HepG2, lung H460, castration-resistant prostate VCaP, gastric SNU-16, and kidney A498 cancer cell lines. The excellent anti-tumor efficacy of 3424 was further demonstrated in PDX models which have high level of AKR1C3 expression, but not in a model with low level of AKR1C3 expression. In the combination therapy, we showed that 3424 could enhance the efficacy of the standard care of chemotherapy in the CDX models. The results described here highlight that 3424 exhibits AKR1C3-dependent cytotoxicity in vitro and anti-tumor activity in vivo in a wide range of human cancer types, which support further development of 3424 as an anti-cancer agent for treating different types of cancers and the use of AKR1C3 as a biomarker to profile cancer patients and further guide patient selection for therapy with 3424.

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers. We report the first crystal structures of the human HDGF PWWP domain (residues 1–100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

AKR1C3 is overexpressed at both levels of protein and RNA in many types of cancer, particularly in liver, gastric, renal, CRPC and non-small cell lung cancer. High AKR1C3 expression is directly related to treatment resistance and poor prognosis. AST-3424/OBI-3424 (denoted by 3424) is a novel prodrug activated by AKR1C3. Currently 3424 is being studied in phase I clinical trials for the treatment of solid and hematologic cancers. Here we characterize 3424 on its specificity against AKR1C3 in biochemical and cell-based assays. In addition, its anti-tumor activity as a single agent or in combination therapy in a broad panel of CDX and PDX models was also investigated. AKR1C3-dependent activation of prodrug 3424 was evident by monitoring the decrease of 3424 and generation of the active form, 2660, based on LC/MS-MS analysis. Kinetic analysis indicated that AKR1C3 exhibited higher catalytic efficiency towards 3424 compared to the physiological substrates. Cytotoxicity of 3424 was found to be dependent on the level of AKR1C3 expression in a wide range of human cancer cell lines. There was a strong correlation between 3424 cytotoxic potency and AKR1C3 expression at both protein and RNA levels across all the cell lines examined. Cytotoxicity of 3424, its enantiomer and racemic mixture were also greatly inhibited by the AKR1C3-specific inhibitor 3021. The racemic mixture induced DNA cross-linking in a concentration dependent manner. Tumor growth inhibition of 3424 was shown to be better than or comparable to the standard of care chemotherapy at clinically achievable doses as a single agent in various CDX models with high expression of AKR1C3 at both levels of protein and RNA, including gastric SNU-16, kidney A498, and castration-resistant prostate VCaP, liver HepG2 and lung H460 cancers. The excellent anti-tumor efficacy of 3424 was further demonstrated in PDX models that have high level of AKR1C3 expression (pancreatic PA1280, gastric GA6201, Lung cancer LU2505) but not in model with low level of AKR1C3 expression (lung cancer LU2057). A significant correlation was found between AKR1C3 expression level and 3424 anti-tumor activity in two lung LU2505 and LU2057 PDX xenograft models confirming that 3424 in vivo activity is AKR1C3-dependent, which is consistent with its AKR1C3-dependent cytotoxicity in vitro. In the combination therapy, we have showed that 3424 could enhance the efficacy of the standard care of chemotherapy in the CDX models of VCaP CRPC, SNU-16 gastric, and A498 renal cell carcinoma. In conclusion, the results described here highlight that 3424 exhibits AKR1C3-dependent cytotoxicity in vitro and anti-tumor activity in vivo in a wide range of human cancer types, which support further development of 3424 as an anti-cancer agent for treating different types of cancer and the use of AKR1C3 as a biomarker to profile cancer patients and further guide patient selection for therapy with 3424. Citation Format: Fanying Meng, Wan-Fen Li, Donald Jung, Chun-Chung Wang, Tianyang Qi, Chi-Sheng Shia, Ren-Yu Hsu, Yin-Cheng Hsieh, Jianxin Duan. Selective and broad anti-tumor activity of AKR1C3-activated prodrug AST-3424/OBI-3424 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1220.

Guan, H., Hsieh, Y., Lin, P. et al. Structural insights into the electron/proton transfer pathways in the quinol : fumarate reductase from Desulfovibrio gigas. Sci Rep 8, 14935 (2018) doi:10.1038/s41598-018-33193-5, The membrane-embedded quinol: fumarate reductase (QFR) in anaerobic bacteria catalyzes the reduction of fumarate to succinate by quinol in the anaerobic respiratory chain. The electron/protontransfer pathways in QFRs remain controversial. Here we report the crystal structure of QFR from the anaerobic sulphate-reducing bacterium Desulfovibrio gigas (D. gigas) at 3.6 Å resolution. The structure of the D. gigas QFR is a homo-dimer, each protomer comprising two hydrophilic subunits, A and B, and one transmembrane subunit C, together with six redox cofactors including two b-hemes. One menaquinone molecule is bound near heme bL in the hydrophobic subunit C. This location of the menaquinone-binding site differs from the menaquinol-binding cavity proposed previously for QFR from Wolinella succinogenes. The observed bound menaquinone might serve as an additional redox cofactor to mediate the proton-coupled electron transport across the membrane. Armed with these structuralinsights, we propose electron/proton-transfer pathways in the quinol reduction of fumarate to succinate in the D. gigas QFR.

Glycosyltransferase-1 from Bacillus cereus (BcGT1) catalyzes a reaction that transfers a glucosyl moiety to flavonoids, such as quercetin, kaempferol, and myricetin. The enzymatic glucosidation shows a broad substrate specificity when the reaction is catalyzed by wild-type BcGT1. Preliminary assays demonstrated that the F240A mutant significantly improves the regioselectivity of enzymatic glucosidation toward quercetin. To unveil and further to control the catalytic function of BcGT1, mutation of F240 to other amino acids, such as C, E, G, R, Y, W, and K, was performed. Among these mutants, F240A, F240G, F240R, and F240K greatly altered the regioselectivity. The quercetin-3-O-glucoside, instead of quercetin-7-O-glucoside as for the wild-type enzyme, was obtained as the major product. Among these mutants, F240R showed nearly 100 % product specificity but only retained 25 % catalytic efficiency of wild-type enzyme. From an inspection of the protein structure, we found two other amino acids, F132 and F138, together with F240, are likely to form a hydrophobic binding region, which is sufficiently spacious to accommodate substrates with varied aromatic moieties. Through the replacement of a phenylalanine by a tyrosine residue in the substrate-binding region, the mutants may be able to fix the orientation of flavonoids, presumably through the formation of a hydrogen bond between substrates and mutants. Multiple mutants-F240R_F132Y, F240R_F138Y, and F240R_F132Y_F138Y-were thus constructed for further investigation. The multiple points of mutants not only maintained the high product specificity but also significantly improved the catalytic efficiency, relative to F240R. The same product specificity was obtained when kaempferol and myricetin were used as a substrate.

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

A novel direct phase-selection method to select optimized phases from the ambiguous phases of a subset of reflections to replace the corresponding initial SAD phases has been developed. With the improved phases, the completeness of built residues of protein molecules is enhanced for efficient structure determination., Optimization of the initial phasing has been a decisive factor in the success of the subsequent electron-density modification, model building and structure determination of biological macromolecules using the single-wavelength anomalous dispersion (SAD) method. Two possible phase solutions (ϕ1 and ϕ2) generated from two symmetric phase triangles in the Harker construction for the SAD method cause the well known phase ambiguity. A novel direct phase-selection method utilizing the θDS list as a criterion to select optimized phases ϕam from ϕ1 or ϕ2 of a subset of reflections with a high percentage of correct phases to replace the corresponding initial SAD phases ϕSAD has been developed. Based on this work, reflections with an angle θDS in the range 35–145° are selected for an optimized improvement, where θDS is the angle between the initial phase ϕSAD and a preliminary density-modification (DM) phase ϕDM NHL. The results show that utilizing the additional direct phase-selection step prior to simple solvent flattening without phase combination using existing DM programs, such as RESOLVE or DM from CCP4, significantly improves the final phases in terms of increased correlation coefficients of electron-density maps and diminished mean phase errors. With the improved phases and density maps from the direct phase-selection method, the completeness of residues of protein molecules built with main chains and side chains is enhanced for efficient structure determination.

Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe 2+ ) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC ( Kp FeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that Kp FeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.

Adenylylsulfate reductase (adenosine 5′-phosphosulfate reductase, APS reductase or APSR, E.C.1.8.99.2) catalyzes the conversion of APS to sulfite in dissimilatory sulfate reduction. APSR was isolated and purified directly from massive anaerobically grown Desulfovibrio gigas, a strict anaerobe, for structure and function investigation. Oligomerization of APSR to form dimers–α_2β_2, tetramers–α_4β_4, hexamers–α_6β_6, and larger oligomers was observed during purification of the protein. Dynamic light scattering and ultracentrifugation revealed that the addition of adenosine monophosphate (AMP) or adenosine 5′-phosphosulfate (APS) disrupts the oligomerization, indicating that AMP or APS binding to the APSR dissociates the inactive hexamers into functional dimers. Treatment of APSR with β-mercaptoethanol decreased the enzyme size from a hexamer to a dimer, probably by disrupting the disulfide Cys156—Cys162 toward the C-terminus of the β-subunit. Alignment of the APSR sequences from D. gigas and A. fulgidus revealed the largest differences in this region of the β-subunit, with the D. gigas APSR containing 16 additional amino acids with the Cys156—Cys162 disulfide. Studies in a pH gradient showed that the diameter of the APSR decreased progressively with acidic pH. To crystallize the APSR for structure determination, we optimized conditions to generate a homogeneous and stable form of APSR by combining dynamic light scattering, ultracentrifugation, and electron paramagnetic resonance methods to analyze the various oligomeric states of the enzyme in varied environments.

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.

Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-d-glucosamine (NAG)n, into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67—Thr-69 and Ile-106–Val-112) to interact with (NAG)n, generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of ∼4.6 Å along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)n reveal (NAG)2, (NAG)2, and (NAG)4 in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)4; in an intermediate state, E145Q/Y227F with a boat-form NAG at the −1 subsite, −1-(NAG); after hydrolysis, E145Q with a chair form −1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between −1-(NAG) and +1-(NAG); Tyr-227 in the conformational change of −1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of −1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu-190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(l-His-l-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)n provide new insights into its substrate binding and the mechanistic action.

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr-I and Dsr-II, are compared at 1.76 and 2.05 A resolution respectively. The dimeric α_2β_2γ_2 structure of Dsr-I contains eight [4Fe–4S] clusters, two saddle-shaped sirohaems and two flat sirohydrochlorins. In Dsr-II, the [4Fe–4S] cluster associated with the sirohaem in Dsr-I is replaced by a [3Fe–4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr-I and Dsr-II confirm the co-factor structures, whereas EPR of a third but inactive form, Dsr-III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe–4S] cluster replaced by a [3Fe–4S] centre. In Dsr-I and Dsr-II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ-subunit C-terminus is inserted into a positively charged channel formed between the α- and β-subunits, with its conserved terminal Cysγ104 side-chain covalently linked to the CHA atom of the sirohaem in Dsr-I. In Dsr-II, the thioether bond is broken, and the Cysγ104 side-chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S_3O_6^(2−), S_2O_3^(2−) and S^(2−).

Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α 2 β 2 -heterotetramer of the Archaeoglobus fulgidus , the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.

Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl -β-acetylthioisobutyrate ( dl -MATI) to produce d -β-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli ; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl -MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67°C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 Å, confirming that EST is a member of the α/β hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ∼12 Å deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl -MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala^(69)–Arg^(74) and Met^(158)–Met^(165), located in the tunnel for the substrate entrance. The substrate/product tunnel is in the “open form” in the apo-TnDhp, in the “intermediate state” in the monometal TnDhp, and in the “closed form” in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala^(69)–Arg^(74) dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine, and N-carbamyl-β-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala(69)-Arg(74) and Met(158)-Met(165), located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the monometal TnDhp, and in the "closed form" in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala(69)-Arg(74) dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine, and N-carbamyl-β-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.

The aminoacylhistidine dipeptidase (PepD) protein encoded by Vibrio alginolyticus pepD was successfully overexpressed and characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The purified enzyme contained two zinc ions per monomer. The recombinant dipeptidase enzyme, which was identified as a homodimer in solution, exhibited broad substrate specificity for Xaa-His dipeptides, with highest activity towards the His-His dipeptide. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Preliminary crystallographic analysis showed that the crystal belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 80.42, c = 303.11 A. The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%.

The branched-chain amino-acid aminotransferase (BCAT), which requires pyridoxal 5'-phosphate (PLP) as a cofactor, is a key enzyme in the biosynthetic pathway of the hydrophobic amino acids leucine, isoleucine and valine. DrBCAT from Deinococcus radiodurans, which has a molecular weight of 40.9 kDa, was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data to 2.50 A resolution from a DrBCAT crystal, the crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.37, b = 90.70, c = 155.47 A. Preliminary analysis indicates the presence of two DrBCAT molecules in the asymmetric unit, with a solvent content of 47.52%.

Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.

Branched-chain aminotransferases (BCAT), which utilize pyridoxal 5′-phosphate (PLP) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three of the most hydrophobic branched-chain amino acids (BCAA), leucine, isoleucine, and valine, to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The BCAT from Deinococcus radiodurans ( Dr BCAT), an extremophile, was cloned and expressed in Escherichia coli for structure and functional studies. The crystal structures of the native Dr BCAT with PLP and its complexes with l -glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The Dr BCAT monomer, comprising 358 amino acids, contains large and small domains connected with an interdomain loop. The cofactor PLP is located at the bottom of the active site pocket between two domains and near the dimer interface. The substrate ( l -glutamate or KIC) is bound with key residues through interactions of the hydrogen bond and the salt bridge near PLP inside the active site pocket. Mutations of some interaction residues, such as Tyr71, Arg145, and Lys202, result in loss of the specific activity of the enzymes. In the interdomain loop, a dynamic loop (Gly173 to Gly179) clearly exhibits open and close conformations in structures of Dr BCAT without and with substrates, respectively. Dr BCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60°C, than either BCAT from Escherichia coli ( e BCAT) or from Thermus thermophilus (HB8BCAT). The dimeric molecular packing and the distribution of cysteine residues at the active site and the molecular surface might explain the resistance to radiation but small thermal stability of Dr BCAT.

The regulatory domain (PA3346RS), comprising the receiver and stalk domains, of the response regulator PA3346 requires phosphorylation for activation with magnesium ions as cofactors in order to modulate the downstream protein phosphatase activity for the regulation of swarming motility in Pseudomonas aeruginosa PAO1. Fusion-tagged recombinant PA3346RS of total molecular mass 25.3 kDa has been overexpressed in Escherichia coli, purified using Ni(2+)-NTA and Q-Sepharose ion-exchange columns and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from PA3346RS crystals to 2.0 Å resolution. The crystal belonged to space group P4(1) or P4(3), with unit-cell parameters a = 82.38, c = 73.34 Å. Preliminary analysis indicated the presence of a dimer of PA3346RS in the asymmetric unit, with a solvent content of 48.6%.

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr-I and Dsr-II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α2β2γ2 structure of Dsr-I contains eight [4Fe-4S] clusters, two saddle-shaped sirohaems and two flat sirohydrochlorins. In Dsr-II, the [4Fe-4S] cluster associated with the sirohaem in Dsr-I is replaced by a [3Fe-4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr-I and Dsr-II confirm the co-factor structures, whereas EPR of a third but inactive form, Dsr-III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe-4S] cluster replaced by a [3Fe-4S] centre. In Dsr-I and Dsr-II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ-subunit C-terminus is inserted into a positively charged channel formed between the α- and β-subunits, with its conserved terminal Cys104 side-chain covalently linked to the CHA atom of the sirohaem in Dsr-I. In Dsr-II, the thioether bond is broken, and the Cys104 side-chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S3O6(2-), S2O3(2-) and S2-.

Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn(2+) at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn(2+)-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn(2+)-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn(2+)-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade beta-propeller fold linked to a C-terminal beta-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (-1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the -1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.

The crystal structure of Bacillus amyloliquefaciens alpha-amylase (BAA) at 1.4 A resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type alpha-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant contribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis alpha-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying alpha-amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.

Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K i value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. Carbamylation on lysine extends the residue length by ~2 Å and changes the side chain from a positive to negative charge at neutral pH. The proteins involved with lysine carbamylation are found to be related to the human diseases, such as type 2 diabetes, developmental delay, metabolic acidosis, mental retardation, hypotonia and seizures. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apo enzyme as well as two forms of the holo enzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified with possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala69–Arg74 and Met158–Met165, located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the mono-metal TnDhp, and in the "closed form" in the di-metal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala69–Arg74 dynamic loop. In addition, the structures of the di-metal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine and N-carbamyl-β-amino isobutyrate, as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.