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ABSTRACT: 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.
International Journal of Molecular Sciences 01/2013; 14(1):1667-83. · 2.60 Impact Factor
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ABSTRACT: Branched-chain aminotransferases (BCAT), which utilize pyridoxal 5' -phosphate (PLP) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three 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 DrBCAT from Deinococcus radiodurans, an extremophile, was cloned and expressed in E. coli for structure and functional studies. The crystal structures of the native DrBCAT with PLP and its complexes with L-glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The DrBCAT 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 DrBCAT without and with substrates, respectively. DrBCAT shows the highest specific activity in both environments of nature and ionizing radiation, but with the less thermal stability above 60 °C, than either BCAT from Escherichia coli (eBCAT) 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 DrBCAT.
Journal of bacteriology 09/2012; · 3.94 Impact Factor
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ABSTRACT: 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%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2011; 67(Pt 8):937-40. · 0.51 Impact Factor
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ABSTRACT: 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–α2β2, tetramers–α4β4, hexamers–α6β6, 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.
04/2011;
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ABSTRACT: 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.
Journal of Biological Chemistry 12/2010; 285(50):39500-10. · 4.77 Impact Factor
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ABSTRACT: 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 PepDCAT. 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 PepDCAT exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepDCAT for GABAergic therapies or neuroprotection.
Journal of Biological Chemistry 12/2010; 285(50):39500-39510. · 4.77 Impact Factor
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ABSTRACT: 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 Zn2+ at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn2+-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 Zn2+-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 Zn2+-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.
Journal of Biological Chemistry 11/2010; 285(48):37872-37883. · 4.77 Impact Factor
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Yin-Cheng Hsieh,
Yue-Jin Wu,
Tzu-Ying Chiang,
Chueh-Yuan Kuo,
Keshab Lal Shrestha,
Cheng-Fu Chao,
Yen-Chieh Huang,
Phimonphan Chuankhayan,
Wen-Guey Wu,
Yaw-Kuen Li,
Chun-Jung Chen
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ABSTRACT: 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.
Journal of Biological Chemistry 10/2010; 285(41):31603-15. · 4.77 Impact Factor
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Yin-Cheng Hsieh,
Yue-Jin Wu,
Tzu-Ying Chiang,
Chueh-Yuan Kuo,
Keshab Lal Shrestha,
Cheng-Fu Chao,
Yen-Chieh Huang,
Phimonphan Chuankhayan,
Wen-guey Wu,
Yaw-Kuen Li,
Chun-Jung Chen
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ABSTRACT: 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.
Journal of Biological Chemistry 10/2010; 285(41):31603-31615. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 10/2010; 285(48):37872-83. · 4.77 Impact Factor
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ABSTRACT: 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-.
Molecular Microbiology 09/2010; 78(5):1101-16. · 5.01 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 05/2010; 285(30):23251-64. · 4.77 Impact Factor
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Jahan Alikhajeh,
Khosro Khajeh,
Bijan Ranjbar,
Hossein Naderi-Manesh,
Yi Hung Lin,
Enhung Liu,
Hong Hsiang Guan, Yin Cheng Hsieh,
Phimonphan Chuankhayan,
Yen Chieh Huang,
Jeyakanthan Jeyaraman,
Ming Yih Liu,
Chun Jung Chen
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ABSTRACT: 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.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2010; 66(Pt 2):121-9. · 0.51 Impact Factor
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ABSTRACT: 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-A resolution. Different from the alpha(2)beta(2)-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six alphabeta-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the alpha-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 alpha-helix (amino acid 289 to 299) in the alpha-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 beta-subunit wraps around the alpha-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the alpha-subunit from another alphabeta-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the alpha-subunit and Asp159 in the C terminus of the beta-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 beta-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 beta-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 beta-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 beta-subunit.
Journal of bacteriology 10/2009; 191(24):7597-608. · 3.94 Impact Factor
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ABSTRACT: 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%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2009; 65(Pt 3):216-8. · 0.51 Impact Factor
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ABSTRACT: 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.
Molecular pharmacology 04/2008; 73(3):660-8. · 4.53 Impact Factor
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ABSTRACT: 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%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2007; 63(Pt 6):492-4. · 0.51 Impact Factor
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ABSTRACT: Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 A resolution, the crystal belongs to space group P2(1), with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 A, beta = 99.31 degrees . Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2006; 62(Pt 9):916-9. · 0.51 Impact Factor
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ABSTRACT: Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-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 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta 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 approximately 12 A 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.
Journal of Bacteriology 01/2006; 187(24):8470-6. · 3.83 Impact Factor
