[Show abstract][Hide abstract] ABSTRACT: Pyridoxal-5'-phosphate (the active form of vitamin B6) is an essential cofactor in many enzymatic reactions. While animals lack any of the pathways for de novo synthesis and salvage of vitamin B6, it is synthesized by two distinct biosynthetic routes in bacteria, fungi, parasites, and plants. One of them is the PdxA/PdxJ pathway found in the gamma subdivision of proteobacteria. It depends on the pdxB gene, which encodes erythronate-4-phosphate dehydrogenase (PdxB), a member of the d-isomer specific 2-hydroxyacid dehydrogenase superfamily. Although three-dimensional structures of other functionally related dehydrogenases are available, no structure of PdxB has been reported. To provide the missing structural information and to gain insights into the catalytic mechanism, we have determined the first crystal structure of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa in the ligand-bound state. It is a homodimeric enzyme consisting of 380-residue subunits. Each subunit consists of three structural domains: the lid domain, the nucleotide-binding domain, and the C-terminal dimerization domain. The latter domain has a unique fold and is largely responsible for dimerization. Interestingly, two subunits of the dimeric enzyme are bound with different combinations of ligands in the crystal and they display significantly different conformations. Subunit A is bound with NAD and a phosphate ion, while subunit B, with a more open active site cleft, is bound with NAD and l(+)-tartrate. Our structural data allow a detailed understanding of cofactor and substrate recognition, thus providing substantial insights into PdxB catalysis.
No preview · Article · Apr 2007 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes
have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane
dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation
of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to
involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone
has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report
the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies
His152 as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.
Preview · Article · Aug 2006 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.
[Show abstract][Hide abstract] ABSTRACT: The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.
Preview · Article · Mar 2006 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
[Show abstract][Hide abstract] ABSTRACT: Agmatine, which results from the decarboxylation of L-arginine by arginine decarboxylase, is a metabolic intermediate in the biosynthesis of putresine and higher polyamines (spermidine and spermine). Recent studies indicate that agmatine can have several important biochemical effects in humans, ranging from effects on the central nervous system to cell proliferation in cancer and viral replication. Agmatinase catalyses the hydrolysis of agmatine to putresine and urea and is a major target for drug action and development. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Human agmatinase (residues Ala36-Val352) has been overexpressed as a fusion with both N- and C-terminal purification tags in Escherichia coli and crystallized in the presence of Mn2+ and 1,6-diaminohexane at 297 K using polyethylene glycol 4000 as a precipitant. X-ray diffraction data were collected at 100 K to 2.49 A from a flash-frozen crystal. The crystals are tetragonal, belonging to space group P4(2), with unit-cell parameters a = b = 114.54, c = 125.65 A, alpha = beta = gamma = 90 degrees. Three monomers are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.66 A3 Da(-1) and a solvent content of 66.4%.
Preview · Article · Nov 2005 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
[Show abstract][Hide abstract] ABSTRACT: Galectin from an edible fungus Agrocybe cylindracea (ACG) has a strong preference for N-acetylneuraminyl lactose (NeuAcalpha2-3lactose). The sugar recognition mechanism of ACG was explored by the X-ray crystallographic analyses of ligand-free ACG, and its complex with lactose, 3'-sulfonyl lactose and NeuAcalpha2-3lactose. The refined structure shows that ACG is a "proto"-type galectin composed of a beta-sandwich of two antiparallel sheets, each with six strands, in contrast to the five and six strands in animal galectins. ACG dimer in solution was classified as being among the "layer"-type. The carbohydrate recognition domain (CRD) of this galectin is common to those of animal galectins, except for substitution of one residue, Ala64, which corresponds to Asn46 in human galectin 1. A five-residue insertion in ACG at positions 42-46 involving Ser44 and Asn46 modified the architecture of the sugar binding site that contributes sialic acid specificity. Furthermore, it was found that the binding of a sulfate ion near the CRD in the ligand-free form led to a change in the conformation of the loop region caused by main-chain cis/trans transition between Ser44 and Pro45.
[Show abstract][Hide abstract] ABSTRACT: The enzyme nicotinic acid mononucleotide adenylyltransferase (NaMN AT; EC 22.214.171.124) is essential for the synthesis of nicotinamide adenine dinucleotide and is a potential target for antibiotics. It catalyzes the transfer of an AMP moiety from ATP to nicotinic acid mononucleotide to form nicotinic acid adenine dinucleotide. In order to provide missing structural information on the substrate complexes of NaMN AT and to assist structure-based design of specific inhibitors for antibacterial discovery, we have determined the crystal structure of NaMN AT from Pseudomonas aeruginosa in three distinct states, i.e. the NaMN-bound form at 1.7A resolution and ATP-bound form at 2.0A as well as its apo-form at 2.0A. They represent crucial structural information necessary for better understanding of the substrate recognition and the catalytic mechanism. The substrate-unbound and substrate-complexed structures are all in the fully open conformation and there is little conformational change upon binding each of the substrates. Our structures indicate that a conformational change is necessary to bring the two substrates closer together for initiating the catalysis. We suggest that such a conformational change likely occurs only after both substrates are simultaneously bound in the active site.
No preview · Article · Sep 2005 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.
No preview · Article · Jul 2005 · Molecules and Cells
[Show abstract][Hide abstract] ABSTRACT: Agmatine is the product of arginine decarboxylation and can be hydrolyzed by agmatinase to putrescine, the precursor for biosynthesis
of higher polyamines, spermidine, and spermine. Besides being an intermediate in polyamine metabolism, recent findings indicate
that agmatine may play important regulatory roles in mammals. Agmatinase is a binuclear manganese metalloenzyme and belongs
to the ureohydrolase superfamily that includes arginase, formiminoglutamase, and proclavaminate amidinohydrolase. Compared
with a wealth of structural information available for arginases, no three-dimensional structure of agmatinase has been reported.
Agmatinase from Deinococcus radiodurans, a 304-residue protein, shows ∼33% of sequence identity to human mitochondrial agmatinase. Here we report the crystal structure
of D. radiodurans agmatinase in Mn2+-free, Mn2+-bound, and Mn2+-inhibitor-bound forms, representing the first structure of agmatinase. It reveals the conservation as well as variation in
folding, oligomerization, and the active site of the ureohydrolase superfamily. D. radiodurans agmatinase exists as a compact homohexamer of 32 symmetry. Its binuclear manganese cluster is highly similar but not identical
to the clusters of arginase and proclavaminate amidinohydrolase. The structure of the inhibited complex reveals that inhibition
by 1,6-diaminohexane arises from the displacement of the metal-bridging water.
Preview · Article · Dec 2004 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Monodehydroascorbate (MDA) radical reductase (EC 126.96.36.199) is an FAD enzyme that catalyzes the univalent reduction of MDA radical to ascorbate using NAD(P)H as an electron donor. The recombinant MDA reductase from cucumber was crystallized using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 60.8, b = 138.6, c = 61.7 A, beta = 114.5 degrees, and contained two molecules per asymmetric unit. The Matthews coefficient (VM) and the solvent content are 2.46 A3 Da(-1) and 50.0%, respectively. Diffraction data were collected to a resolution of 2.4 A at 100 K using Cu Kalpha radiation with a multi-wire area detector and gave a data set with an overall Rsym of 10.0% and a completeness of 92.5%.
No preview · Article · Sep 2004 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: The enzyme nicotinic acid mononucleotide adenylyltransferase (NaMN AT; EC 188.8.131.52) is essential for the synthesis of nicotinamide adenine dinucleotide and is a potential target for antibiotics. It catalyzes the transfer of an adenyl group from ATP to nicotinic acid mononucleotide to form nicotinic acid adenine dinucleotide. NaMN AT from Pseudomonas aeruginosa was overexpressed in Escherichia coli and crystallized at 291 K using 100 mM bis-Tris propane pH 7.0, 700 mM trisodium citrate and 15%(v/v) glycerol. X-ray diffraction data have been collected to 1.70 A. The crystals are tetragonal, belonging to space group P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 65.02, c = 109.80 A. The presence of one monomer in the asymmetric unit gives a reasonable V(M) of 2.15 A(3) Da(-1), with a solvent content of 42.7%.
No preview · Article · Jun 2004 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: The RecR protein plays a key role in the RecFOR pathway of recombination, which is necessary for the repair of ssDNA gaps. RecR from Deinococcus radiodurans has been overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 1000 as a precipitant. X-ray diffraction data to 2.90 A resolution have been collected at 100 K using Cu Kalpha X-rays from a mercury-soaked crystal. The crystal belongs to space group C222(1), with unit-cell parameters a = 106.96, b = 122.25, c = 156.01 A. The asymmetric unit contains four monomers of RecR, with a crystal volume per protein weight (V(M)) of 2.57 A(3) Da(-1) and a solvent content of 51.0%.
No preview · Article · Mar 2004 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate to chorismate in the shikimate pathway, which represents an attractive target for discovering antimicrobial agents and herbicides. Chorismate serves as a common precursor for the synthesis of aromatic amino acids and many aromatic compounds in microorganisms and plants. Chorismate synthase requires reduced FMN as a cofactor but the catalyzed reaction involves no net redox change. Here, we have determined the crystal structure of chorismate synthase from Helicobacter pylori in both FMN-bound and FMN-free forms. It is a tetrameric enzyme, with each monomer possessing a novel "beta-alpha-beta sandwich fold". Highly conserved regions, including several flexible loops, cluster together around the bound FMN to form the active site. The unique FMN-binding site is formed largely by a single subunit, with a small contribution from a neighboring subunit. The isoalloxazine ring of the bound FMN is significantly non-planar. Our structure illuminates the essential functional roles played by the cofactor.
Preview · Article · Mar 2004 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The enzyme HemK (or PrmC) is one of the first identified methyltransferases that modify glutamine. It methylates the highly conserved GGQ motif in class I release factors (RF1 and RF2) in Escherichia coli. HemK from Thermotoga maritima was over-expressed and crystallized in the presence of S-adenosylmethionine at 296 K using ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystal is orthorhombic, belonging to the space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters of a = 104.24, b = 118.73, and c = 146.62 A. Two (or three) monomers of recombinant HemK are likely to be present in the crystallographic asymmetric unit, giving a V(M) of 3.62 A3 Da(-1) (or 2.41 A3 Da(-1)), with a solvent content of 62.7% (or 44.0%).
No preview · Article · Nov 2003 · Molecules and Cells
[Show abstract][Hide abstract] ABSTRACT: tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to G(37) within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G(36)G(37) in the active site of TrmD and the catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD(2)-tRNA(2) complex, which provides insights into recognition of the general tRNA structure by TrmD.
[Show abstract][Hide abstract] ABSTRACT: Acetohydroxy acid isomeroreductase (AHIR) is a key enzyme in the biosynthesis of branched-chain amino acids. We have determined the first crystal structure of a class I AHIR from Pseudomonas aeruginosa at 2.0 A resolution. Its dodecameric architecture of 23 point group symmetry is assembled of six dimeric units and dimerization is essential for the formation of the active site. The dimeric unit of P.aeruginosa AHIR partially superimposes with a three-domain monomer of spinach AHIR, a class II enzyme. This demonstrates that the so-called plant-specific insert in the middle of spinach AHIR is structurally and functionally equivalent to the C-terminal alpha-helical domain of P.aeruginosa AHIR, and the C-terminal alpha-helical domain was duplicated during evolution from the shorter, class I AHIRs to the longer, class II AHIRs. The dimeric unit of P.aeruginosa AHIR possesses a deep figure-of-eight knot, essentially identical with that in the spinach AHIR monomer. Thus, our work lowers the likelihood of the previous proposal that "domain duplication followed by exchange of a secondary structure element can be a source of such a knot in the protein structure" being correct.
No preview · Article · May 2003 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The enzyme tRNA(m(1)G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) specifically to guanosine at position 37 within a subset of tRNA species in bacteria. The modified guanosine is next to the anticodon and is important for the maintenance of the correct reading frame during translation. TrmD from Haemophilus influenzae with both N- and C-terminal tags was overexpressed in Escherichia coli and crystallized at 297 K using sodium acetate as a precipitant. Native X-ray diffraction data were collected to 1.85 A resolution. The crystals are rhombohedral, belonging to the space group R32, with unit-cell parameters a = b = 98.05, c = 176.79 A, alpha = beta = 90, gamma = 120 degrees. The presence of one monomer of recombinant TrmD in the crystallographic asymmetric unit gives a V(M) of 3.07 A(3) Da(-1) and a solvent content of 59.9%.
No preview · Article · Feb 2003 · Acta Crystallographica Section D Biological Crystallography
[Show abstract][Hide abstract] ABSTRACT: The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-manno-octulosonate (KDO) by forming CMP-KDO. It is essential for the biosynthesis of lipopolysaccharides in Gram-negative bacteria and is a potential target for the discovery of antibacterial agents. L-CKS from Haemophilus influenzae was overexpressed with a C-terminal hexahistidine tag in Escherichia coli and crystallized in the presence of the substrate KDO at 297 K using PEG 4000 as a precipitant and ethylene glycol as an additive. The diffraction limit and spot shape of the native crystal could be improved significantly by dehydration/annealing. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 83.1, c = 117.3 A. The presence of two monomers of recombinant L-CKS in the crystallographic asymmetric unit gives a reasonable V(M) of 2.05 A(3) Da(-1), with a solvent content of 40.0%.
No preview · Article · Feb 2003 · Acta Crystallographica Section D Biological Crystallography