Browsing by Author "Chen, Chun-Jung"

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Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases
(2010-12-01)
Chang, Chin-Yuan
;
Hsieh, Yin-Cheng
;
Wang, Ting-Yi
;
Chen, Yi-Chin
;
Wang, Yu-Kuo
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 gamma-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.
Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases
(2010-12-01)
Chang, Chin-Yuan
;
Hsieh, Yin-Cheng
;
Wang, Ting-Yi
;
Chen, Yi-Chin
;
Wang, Yu-Kuo
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 gamma-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.
Crystal structure of a secondary vitamin D(3) binding site of milk beta-lactoglobulin
(2008-05-15)
Yang, Ming-Chi
;
Guan, Hong-Hsiang
;
Liu, Ming-Yih
;
Lin, Yih-Hung
;
Yang, Jinn-Moon
beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 angstrom resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 angstrom). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an a-helix and a beta-strand I with 17.91 angstrom in length, while the span of vitamin D(3) is about 12.51 angstrom. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure Of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).
Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 angstrom resolution
(2006-10-13)
Chen, Chun-Jung
;
Lin, Yi-Hung
;
Huang, Yen-Chieh
;
Liu, Ming-Yih
;
生物科技學系
Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin: oxygen oxidoreductase (ROO) in D.g. The crystal structure of D.g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 angstrom resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and it pi-pi interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N-H(...)SG of the iron-sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C-O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultra-high-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur. cluster, and distributions of electron density among activity sites. For the first time, this information will provide a clear role for this protein in a strict anaerobic bacterium. (c) 2006 Elsevier Inc. All rights reserved.
Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS
(2010-10-08)
Hsieh, Yin-Cheng
;
Wu, Yue-Jin
;
Chiang, Tzu-Ying
;
Kuo, Chueh-Yuan
;
Shrestha, Keshab Lal
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 similar to 4.6 angstrom 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, Glu190, 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.
Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS
(2010-10-08)
Hsieh, Yin-Cheng
;
Wu, Yue-Jin
;
Chiang, Tzu-Ying
;
Kuo, Chueh-Yuan
;
Shrestha, Keshab Lal
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 similar to 4.6 angstrom 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, Glu190, 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.
Crystal Structures of Vertebrate Dihydropyrimidinase and Complexes from Tetraodon nigroviridis with Lysine Carbamylation METAL AND STRUCTURAL REQUIREMENTS FOR POST-TRANSLATIONAL MODIFICATION AND FUNCTION
(2013-10-18)
Hsieh, Yin-Cheng
;
Chen, Mei-Chun
;
Hsu, Ching-Chen
;
Chan, Sunney I.
;
Yang, Yuh-Shyong
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--alanine, and N-carbamyl--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.
Evidence for beta-lactoglobulin involvement in vitamin D transport in vivo- role of the gamma-turn (Leu-Pro-Met) of beta-lactoglobulin in vitamin D binding
(2009-04-01)
Yang, Ming Chi
;
Chen, Nai Chi
;
Chen, Chun-Jung
;
Wu, Chin Yun
;
Mao, Simon J. T.
beta-lactoglobulin (LG) is a major bovine milk protein, containing a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Crystallographic findings from our previous study showed the exosite to be located at the pocket between the alpha-helix and beta-strand I. In the present study, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the gamma-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D(3) to block the binding of a specific mAb in the gamma-turn loop. Using the mouse (n = 95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D(3) revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2 : 1 (vitamin D(3)/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1 : 1. We have demonstrated for the first time that LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake.
Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)
(2008-03-01)
Lu, Lu-Yi
;
Hsieh, Yin-Cheng
;
Liu, Ming-Yih
;
Lin, Yih-Hung
;
Chen, Chun-Jung
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.
Purification, crystallization and preliminary X-ray analysis of an aminoacylhistidine dipeptidase (PepD) from Vibrio alginolyticus
(2009-03-01)
Chang, Chin-Yuan
;
Hsieh, Yin-Cheng
;
Wang, Ting-Yi
;
Chen, Chun-Jung
;
Wu, Tung-Kung
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 angstrom. The crystal contained two molecules per asymmetric unit and the predicted solvent content was 53.4%.
Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2
(2006-09-01)
Kuo, Chueh-Yuan
;
Wu, Yue-Jin
;
Hsieh, Yin-Cheng
;
Guan, Hong-Hsiang
;
Tsai, Huei-Ju
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 angstrom resolution, the crystal belongs to space group P2(1), with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 angstrom, beta = 99.31 degrees. Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.
Purification, crystallization and preliminary X-ray crystallographic analysis of glycosyltransferase-1 from Bacillus cereus
(2014-09-01)
Hsieh, Yin-Cheng
;
Chiu, Hsi-Ho
;
Huang, Yen-Chieh
;
Fun, Hoong-Kun
;
Lu, Chia-Yu
Glycosyltransferases (GTs), which are distributed widely in various organisms, including bacteria, fungi, plants and animals, play a role in synthesizing biological compounds. Glycosyltransferase-1 from Bacillus cereus (BcGT-1), which is capable of transferring glucose to small molecules such as kaempferol and quercetin, has been identified as a member of the family 1 glycosyltransferases which utilize uridine diphosphate glucose (UDP-glucose) as the sugar donor. BcGT-1 (molecular mass 45.5 kDa) has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction of BcGT-1 crystals to 2.10 angstrom resolution, the crystal belonged to space group P1, with unit-cell parameters a = 54.56, b = 84.81, c = 100.12 A, alpha = 78.36, beta = 84.66, gamma = 84.84 degrees. Preliminary analysis indicates the presence of four BcGT-1 molecules in the asymmetric unit with a solvent content of 50.27%.
Purification, crystallization and preliminary X-ray crystallographic analysis of xylose reductase from Candida tropicalis
(2009-04-01)
Chen, Li-Chun
;
Huang, Sheng-Cih
;
Chuankhayan, Phimonphan
;
Chen, Chung-Der
;
Huang, Yen-Chieh
Xylose reductase (XR), which requires NADPH as a co-substrate, catalyzes the reduction of D-xylose to xylitol, which is the first step in the metabolism of D-xylose. The detailed three-dimensional structure of XR will provide a better understanding of the biological significance of XR in the efficient production of xylitol from biomass. XR of molecular mass 36.6 kDa from Candida tropicalis was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data from C. tropicalis XR crystals at 2.91 angstrom resolution, the unit cell belongs to space group P3(1) or P3(2). Preliminary analysis indicated the presence of four XR molecules in the asymmetric unit, with 68.0% solvent content.
Rational crystallization of beta -lactoglobulin and vitamin D-3 complex reveal a secondary binding site
(2008-01-01)
Chen, Chun-Jung
;
Guan, Hong-Hsiang
;
Yang, Ming-Chi
;
Moo, Simon J. T.
;
生物科技學系
Rational Design for Crystallization of beta-Lactoglobulin and Vitamin D(3) Complex: Revealing a Secondary Binding Site
(2008-12-01)
Yang, Ming Chi
;
Guan, Hong-Hsiang
;
Yang, Jirin-Moon
;
Ko, Cheng-Neng
;
Liu, Ming-Yih
beta-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D(3) binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D3 in the complex by altering the pH and initial ratios of vitamin D(3)/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D(3)/LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D(3) in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.
Single femtosecond laser pulse-single crystal formation of glycine at the solution surface
(2013-03-01)
Liu, Tsung-Han
;
Uwada, Takayuki
;
Sugiyama, Teruki
;
Usman, Anwar
;
Hosokawa, Yoichiroh
We demonstrate femtosecond laser-induced crystallization of glycine from its supersaturated solution depending on laser tunable parameters (pulse energy and repetition rate) and focal position, and examine the crystallization probability, crystal morphology, and crystal polymorph. The generation of cavitation bubble through multiphoton absorption of water depends on input laser pulse energy and repetition rate, which strongly determine morphology and number of the obtained crystals. Significant increase in the crystallization probability is observed by irradiating the femtosecond laser pulses to the air/solution interface, and single pulse-induced single crystal formation is successfully achieved. The crystallization mechanism is discussed in view of inhomogeneous mechanical stress induced by cavitation bubble generation and molecular assembly characteristics of the surface. (C) 2012 Elsevier B.V. All rights reserved.
Structural insights into the histidine-containing phosphotranster protein and receiver domain of sensor histidine kinase suggest a complex model in the two-component regulatory system in Pseudomonas aeruginosa
(2020-09-01)
Chen, Shao-Kang
;
Guan, Hong-Hsiang
;
Wu, Pei-Hsun
;
Lin, Li-Ting
;
Wu, Meng-Chun
In Pseudomonas aeruginosa, an important opportunistic pathogen that causes numerous acute and chronic infections, the hybrid two-component system (TCS) regulates the swarming ability and biofilm formation with a multistep phosphorelay, and consists of hybrid-sensor histidine kinase (HK), histidine-containing phosphotransfer protein (Hpt) and response regulator (RR). In this work, two crystal structures of HptB and the receiver domain of HK PA1611 (PA1611REC) of P. aeruginosa have been determined in order to elucidate their interactions for the transfer of the phosphoryl group. The structure of HptB folds into an elongated four-helix bundle - helices alpha 2, alpha 3, alpha 4 and alpha 5, covered by the short N-terminal helix alpha 1. The imidazole side chain of the conserved activesite histidine residue His57, located near the middle of helix alpha 3, protrudes from the bundle and is exposed to solvent. The structure of PA1611REC possesses a conventional (beta/alpha)(5) topology with five-stranded parallel beta-sheets folded in the central region, surrounded by five alpha-helices. The divalent Mg2+ ion is located in the negatively charged active-site cleft and interacts with Asp522, Asp565 and Arg567. The HptB-PA1611REC complex is further modeled to analyze the binding surface and interactions between the two proteins. The model shows a shape complementarity between the convex surface of PA1611REC and the kidney-shaped HptB with fewer residues and a different network involved in interactions compared with other TCS complexes, such as SLN1-R1/YPD1 from Saccharomyces cerevisiae and AHK5(RD)/AHP1 from Arabidopsis thaliana. These structural results provide a better understanding of the TCS in P. aeruginosa and could potentially lead to the discovery of a new treatment for infection.
Structure of a Secondary Vitamin D-3 Binding Site of Milk beta-Lactoglobulin for Vitamin D Transport.
(2009-01-01)
Chen, Chun-Jung
;
Yang, Ming-Chi
;
Guan, Hong-Hsiang
;
Yang, Jinn-Moon
;
Chen, Wen-Liang
Three important amino acids control the regioselectivity of flavonoid glucosidation in glycosyltransferase-1 from Bacillus cereus
(2016-10)
Chiu, Hsi-Ho
;
Hsieh, Yin-Cheng
;
Chen, Ya-Huei
;
Wang, Hsin-Ying
;
Lu, Chia-Yu
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.