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1.  Structure, Dynamics, Evolution, and Function of a Major Scaffold Component in the Nuclear Pore Complex 
Summary
The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC’s inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2–960 [ScNup192(2–960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2–960) could undergo long-range transition between “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2–960), and homology modeling. Evolutionary analyses using the ScNup192(2–960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.
doi:10.1016/j.str.2013.02.005
PMCID: PMC3755625  PMID: 23499021
2.  Structure of alanine racemase from Oenococcus oeni with bound pyridoxal 5′-phosphate 
Alanine racemase from O. oeni exists as a dimer in the crystal structure. Both monomers contribute to the two active sites present, one for each monomer.
The crystal structure of alanine racemase from Oenococcus oeni has been determined at 1.7 Å resolution using the single-wavelength anomalous dispersion (SAD) method and selenium-labelled protein. The protein exists as a symmetric dimer in the crystal, with both protomers contributing to the two active sites. Pyridoxal 5′-phosphate, a cofactor, is bound to each monomer and forms a Schiff base with Lys39. Structural comparison of alanine racemase from O. oeni (Alr) with homologous family members revealed similar domain organization and cofactor binding.
doi:10.1107/S1744309112047276
PMCID: PMC3539696  PMID: 23295479
alanine racemase; Oenococcus oeni; pyridoxal 5′-phosphate
3.  Structure of a periplasmic glucose-binding protein from Thermotoga maritima  
The periplasmic glucose-binding protein from T. maritima consists of two domains with the ligand β-d-glucose buried between them. The two domains adopt a closed conformation.
ABC transport systems have been characterized in organisms ranging from bacteria to humans. In most bacterial systems, the periplasmic component is the primary determinant of specificity of the transport complex as a whole. Here, the X-ray crystal structure of a periplasmic glucose-binding protein (GBP) from Thermotoga maritima determined at 2.4 Å resolution is reported. The molecule consists of two similar α/β domains connected by a three-stranded hinge region. In the current structure, a ligand (β-d-glucose) is buried between the two domains, which have adopted a closed conformation. Details of the substrate-binding sites revealed features that determine substrate specificity. In toto, ten residues from both domains form eight hydrogen bonds to the bound sugar and four aromatic residues (two from each domain) stabilize the substrate through stacking interactions.
doi:10.1107/S1744309112045241
PMCID: PMC3509965  PMID: 23192024
glucose-binding proteins; ABC transporters; hinge motion; closed conformation
4.  1-Methylthio-D-Xylulose 5-Phosphate Methylsulfurylase: A Novel Route to 1-Deoxy-D-Xylulose 5-Phosphate in Rhodospirillum rubrum 
Biochemistry  2012;51(42):8324-8326.
Rhodospirillum rubrumproduces 5-methylthioadenosine (MTA) from S-adenosylmethionine (SAM) in polyamine biosynthesis; however, R. rubrum lacks the classical methionine salvage pathway. Instead, MTA is converted to 5-methylthio-D-ribose 1-phosphate (MTR 1-P) and adenine; MTR 1-P is isomerized to 1-methylthio-D-xylulose 5-phosphate (MTXu 5-P) and reductively dethiomethylated to 1-deoxy-D-xylulose 5-phosphate (DXP), an intermediate in the nonmevalonate isoprenoid pathway (Erb et al. Nature Chem. Biol., in press). Dethiomethylation, a novel route to DXP, is catalyzed by MTXu 5-P methylsulfurylase. An active site Cys displaces the enolate of DXP from MTXu 5-P, generating a methyl disulfide intermediate.
doi:10.1021/bi301215g
PMCID: PMC3490199  PMID: 23035785
5.  Structure and Catalytic Mechanism of LigI: Insight into the Amidohydrolase Enzymes of cog3618 and Lignin Degradation† 
Biochemistry  2012;51(16):3497-3507.
LigI from Sphingomonas paucimobilis catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. This protein is a member of the amidohydrolase superfamily of enzymes. The protein was expressed in E. coli and then purified to homogeneity. The purified recombinant enzyme does not contain bound metal ions and the addition of metal chelators or divalent metal ions to the assay mixtures does not affect the rate of product formation. This is the first enzyme from the amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. The kinetic constants for the hydrolysis of PDC are 340 s−1 and 9.8 × 106 M−1s−1 for the values of kcat, and kcat/Km respectively. The pH dependence on the kinetic constants suggests that a single active site residue must be deprotonated for the hydrolysis of PDC. The site of nucleophilic attack was determined by conducting the hydrolysis of PDC in 18O-labeled water and subsequent 13C NMR analysis. The crystal structures of wild-type LigI and the D248A mutant in the presence of the reaction product were determined to a resolution of 1.9 Å. The C-8 and C-11 carboxylate groups of PDC are coordinated within the active site via ion pair interactions with Arg-130 and Arg-124, respectively. The hydrolytic water molecule is activated by a proton transfer to Asp-248. The carbonyl group of the lactone substrate is activated by electrostatic interactions with His-180, His-31 and His-33.
doi:10.1021/bi300307b
PMCID: PMC3416963  PMID: 22475079
6.  Crystal structure of a putative lysostaphin peptidase from Vibrio cholerae 
Proteins  2008;72(3):1096-1103.
doi:10.1002/prot.22095
PMCID: PMC3614409  PMID: 18498110
lysostaphin peptidase; LytM; glycyl-glycine or glycyl-alanine; latent form
7.  Structural Insight into Mechanism and Diverse Substrate Selection Strategy of L-Ribulokinase 
Proteins  2011;80(1):261-268.
The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyses conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyses phosphorylation of all four 2-ketopentose sugars with comparable kcat values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptide chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified 447GGLPQK452 as the ATP binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the sidechains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases, revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.
doi:10.1002/prot.23202
PMCID: PMC3240725  PMID: 22072612
Crystal structure; ribulokinase; ribulose; araBAD; araB; arabinose-catalolism
8.  Pa0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine† 
Biochemistry  2011;50(30):6589-6597.
Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g, have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with values of kcat/Km that exceed 105 M−1s−1. These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted (β/α)8-barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine and hypoxanthine were also determined thereby permitting identification of the residues responsible for coordinating the substrate and product.
doi:10.1021/bi200868u
PMCID: PMC3151671  PMID: 21710971
9.  Protein-RNA and Protein-Protein Recognition by Dual KH1/2 Domains of the Neuronal Splicing Factor Nova-1 
Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We report the crystal structure of the first two KH domains (KH1/2) of Nova-1 bound to an in vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site. Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. While the canonical RNA-binding surface of KH2 in the above complex engages in protein-protein interactions in the crystalline state, the individual KH2 domain can sequence-specifically target the UCAC RNA element in solution. The observed anti-parallel alignment of KH1 and KH2 domains in the crystal structure of the complex generates a scaffold that could facilitate target pre-mRNA looping upon Nova binding, thereby potentially explaining Nova’s functional role in splicing regulation.
doi:10.1016/j.str.2011.05.002
PMCID: PMC3134789  PMID: 21742260
11.  Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase† 
Biochemistry  2011;50(11):1917-1927.
Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (kcat = 2.0 s−1; kcat/Km = 2.5 × 103 M−1 s−1). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with values of kcat and kcat/Km of 200 s−1 and 5 × 105 M−1s−1, respectively. The apo-enzyme was prepared and reconstituted with Fe2+, Zn2+, or Mn2+. In each case, two enzyme-equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member within the deaminase sub-family of the amidohydrolase superfamily (AHS) to utilize a binuclear metal center for the catalysis of a deamination reaction. [FeII/FeII]-ADE was oxidized to [FeIII/FeIII]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [FeIII/FeIII]-ADE with dithionite restored the deaminase activity and thus the di-ferrous form of the enzyme is essential for catalytic activity. No evidence for spin-coupling between metal ions was evident by EPR or Mössbauer spectroscopies. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 Å resolution and adenine was modeled into the active site based on homology to other members of the amidohydrolase superfamily. Based on the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH rate profiles and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate limiting steps.
doi:10.1021/bi101788n
PMCID: PMC3059353  PMID: 21247091
12.  Enzymatic Deamination of the Epigenetic Base N-6-Methyladenine 
Two enzymes of unknown function from the amidohydrolase superfamily were discovered to catalyze the deamination of N-6-methyladenine to hypoxanthine and methyl amine. The methylation of adenine in bacterial DNA is a common modification for the protection of host DNA against restriction endonucleases. The enzyme from Bacillus halodurans, Bh0637, catalyzes the deamination of N-6-methyladenine with a kcat of 185 s−1 and a kcat/Km of 2.5 × 106 M−1 s−1. Bh0637 catalyzes the deamination of N-6-methyladenine two orders of magnitude faster than adenine. A comparative model of Bh0637 was computed using the three-dimensional structure of Atu4426 (PDB code: 3NQB) as a structural template and computational docking was used to rationalize the preferential utilization of N-6-methyladenine over adenine. This is the first identification of an N-6-methyladenine deaminase (6-MAD).
doi:10.1021/ja110157u
PMCID: PMC3043370  PMID: 21275375
13.  Structural and Functional Studies of Fatty Acyl-Adenylate Ligases from E. coli and L. pneumophila 
Journal of molecular biology  2010;406(2):313-324.
Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis (Mtb). They are similar in sequence to fatty acyl-CoA ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full length FAALs from E. coli (EcFAAL) and Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 Å, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, whereas they preclude CoA binding thereby preventing CoA ligation.
doi:10.1016/j.jmb.2010.12.011
PMCID: PMC3040979  PMID: 21185305
Fatty acyl-AMP ligase; Fatty acyl-CoA ligase; X-ray structure; AMP; CoA
14.  A common catalytic mechanism for proteins of HutI family† 
Biochemistry  2008;47(20):5608-5615.
Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase; EC 3.5.2.7) is a member of amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino -L-glutamate in the histidine degradation pathway. We have determined the three dimensional crystal structures of HutI from A. tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [N-formimino-L-glutamate (NIG)] and an inhibitor [3-(2,5-dioxoimidazolidin-4yl)-propionic acid (DIP), respectively. In both structures the active site is contained within each monomer and its organization displays the landmark feature of amidohydrolase superfamily showing a metal ligand (iron), four histidines and one aspartic acid. A catalytic mechanism involving His265 is proposed based on the inhibitor bound structure. This mechanism is applicable to all HutI.
doi:10.1021/bi800180g
PMCID: PMC3232013  PMID: 18442260
AHS; amidohydrolases; NIG; DIP; At-HutI; Es-HutI
15.  Structural studies on cytosolic domain of Magnesium transporter MgtE from Enterococcus faecalis 
Proteins  2010;78(2):487-491.
doi:10.1002/prot.22585
PMCID: PMC3221319  PMID: 19787770
Magnesium transporter; cytosolic domain; x-ray structure
16.  X-ray crystal structure of the B component of Hemolysin BL from Bacillus cereus 
Proteins  2008;71(2):534-540.
Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.
doi:10.1002/prot.21888
PMCID: PMC3220058  PMID: 18175317
HBL-B; Hemolysin; pore-formation; β-hairpain
17.  Mass Spectrometry Guided In Situ Proteolysis to Obtain Crystals for X-ray Structure Determination 
A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration. We report our experience with ten proteins of unknown structure, two of which yielded high-resolution X-ray structures. The advantage of this approach comes from its ability to select only those structure determination candidates that are likely to benefit from application of in situ proteolysis, using conditions most likely to result in formation of a stable proteolytic digestion product suitable for crystallization.
doi:10.1016/j.jasms.2010.06.015
PMCID: PMC2963156  PMID: 20685133
Mass spectrometry; in situ proteolysis; crystallization; x-ray crystallography
18.  Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily 
Biochemistry  2010;49(31):6791-6803.
Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of L-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-L-Pro, L-Ala-L-Pro and N-acyl derivatives of L-Pro. The best substrate identified to date is N-acetyl-L-Pro with a value of kcat/Km of 3 × 105 M−1 s−1. Sgx9260b catalyzes the hydrolysis of L-hydrophobic L-Pro dipeptides and N-acyl derivatives of L-Pro. The best substrate identified to date is N-propionyl-L-Pro with a value of kcat/Km of 1 × 105 M−1 s−1. Three dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes: 3MKV and 3FEQ). These proteins fold as distorted (β/α)8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methyl phosphonate derivative of L-Pro (PDB code: 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center and one oxygen atom interacts with His-140. The α-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows β-strand 7 within the (β/α)8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methyl phosphonate derivative of L-arginine (PDB code: 3MTW).
doi:10.1021/bi100897u
PMCID: PMC2914802  PMID: 20604542
19.  Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase† 
Biochemistry  2010;49(20):4374-4382.
Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin-6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 Å resolution (PDB code: 2PAJ). This protein folds as a distorted (β/α)8-barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosyl homocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin-6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s−1, 8.0 μM, and 1.3 × 105 M−1 s−1 for kcat, Km, and kcat/Km, respectively. The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (PDB code: 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed based upon the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that hydrogen bond with the carbonyl oxygen at C4, a conserved threonine residue that hydrogen bonds with N5, and another conserved threonine residue that hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin.
doi:10.1021/bi100252s
PMCID: PMC2892419  PMID: 20415463
20.  Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR 
Structure (London, England : 1993)  2010;18(11):1512-1521.
SUMMARY
Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a novel family of 2OG-sensing proteins – the nitrogen regulatory protein NrpR – that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR re-positions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
doi:10.1016/j.str.2010.08.014
PMCID: PMC2996049  PMID: 21070950
Nitrogen assimilation; Nitrogen regulation; Electron microscopy; X-ray crystallography; 2-oxoglutarate (2OG); α-ketoglutarate; transcriptional regulation
21.  The Hunt for 8-Oxoguanine Deaminase 
An enzyme from Pseudomonas aeruginosa, Pa0142 (gi|9945972) has been identified for the first time that is able to catalyze the deamination of 8-oxoguanine (8-oxoG) to uric acid. 8-Oxoguanine is formed by the oxidation of guanine residues within DNA by reactive oxygen species and this lesion results in the G:C to T:A transversions. The value of kcat/Km for the deamination of 8-oxoG by Pa0142 at pH 8.0 and 30 °C is 2.0 × 104 M−1 s−1. This enzyme can also catalyze the deamination of isocystosine and guanine at rates that are approximately an order of magnitude slower. The three-dimensional structure of a homologous enzyme (gi|44264246) from the Sargasso Sea has been determined by x-ray diffraction methods to a resolution of 2.2Å (PDB code: 3h4u). The enzyme folds as a (β/α)8− barrel and it is a member of the amidohydrolase superfamily with a single zinc in the active site. This enzyme catalyzes the deamination of 8-oxoG with a value of kcat/Km of 2.7 × 105 M−1 s−1. Computational docking of potential high energy intermediates for the deamination reaction to the x-ray crystal structure suggests that the active site binding of 8-oxoG is facilitated by hydrogen bond interactions from a conserved glutamine that follows β-strand 1 with O6, a conserved tyrosine that follows β-strand 2 with N7, and a conserved cysteine residue that follows β-strand 4 with O8. A bioinformatic analysis of available protein sequences suggest that approximately 200 other bacteria possess an enzyme capable of catalyzing the deamination of 8-oxoG.
doi:10.1021/ja909817d
PMCID: PMC2820149  PMID: 20088583
22.  Structure of YqgQ protein from Bacillus subtilis, a conserved hypothetical protein 
The crystal structure of the hypothetical protein YqgQ from B. subtilis is reported at 2.1 Å resolution.
The crystal structure of the hypothetical protein YqgQ from Bacillus subtilis has been determined to 2.1 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 51.85, b = 41.25, c = 55.18 Å, β = 113.4°, and contained three protein molecules in the asymmetric unit. The structure was determined by the single-wavelength anomalous dispersion method using selenium-labeled protein and was refined to a final R factor of 24.7% (R free = 28.0%). The protein molecule mainly comprises a three-helical bundle. Its putative function is inferred to be single-stranded nucleic acid binding based on sequence and structural homology.
doi:10.1107/S1744309109047009
PMCID: PMC2805524  PMID: 20057058
YqgQ hypothetical protein; Se-SAD; novel fold; DUF910; Protein Structure Initiative; New York SGX Research Consortium for Structural Genomics
23.  Computation-Facilitated Assignment of Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis† 
Biochemistry  2009;48(48):11546-11558.
The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI: 23100298; IMG locus tag Ob2843; PDB Code 2OQY) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg2+ ions located 10.4 Å from one another, with one located in the canonical position in the (β/α)7β-barrel domain (although the ligand at the end of the fifth β-strand is His, unprecedented in structurally characterized members of the superfamily); the second is located in a novel site within the capping domain. In silico docking of a library of mono- and diacid sugars to the active site predicted a diacid sugar as a likely substrate. Activity screening of a physical library of acid sugars identified galactarate as the substrate (kcat = 6.8 s−1, KM = 620 μM; kcat/KM = 1.1 × 104 M−1 s−1), allowing functional assignment of Ob2843 as galactarate dehydratase (GalrD-II) The structure of a complex of the catalytically impaired Y90F mutant with Mg2+ and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the α-proton and Tyr 90 as the acid that facilitates departure of the β-OH leaving group. The enzyme product is 2-keto-3-deoxy-D-threo-4,5-dihydroxyadipate, the enantiomer of the product obtained in the GalrD reaction catalyzed by a previously characterized bifunctional L-talarate/galactarate dehydratase (TalrD/GalrD). On the basis of the different active site structures and different regiochemistries, we recognize that these functions represent an example of apparent, not actual, convergent evolution of function. The structure of GalrD-II and its active site architecture allow identification of the seventh functionally and structurally characterized subgroup in the enolase superfamily. This study provides an additional example that an integrated sequence/structure-based strategy employing computational approaches is a viable approach for directing functional assignment of unknown enzymes discovered in genome projects.
doi:10.1021/bi901731c
PMCID: PMC2787699  PMID: 19883118
24.  ModBase, a database of annotated comparative protein structure models, and associated resources 
Nucleic Acids Research  2010;39(Database issue):D465-D474.
ModBase (http://salilab.org/modbase) is a database of annotated comparative protein structure models. The models are calculated by ModPipe, an automated modeling pipeline that relies primarily on Modeller for fold assignment, sequence–structure alignment, model building and model assessment (http://salilab.org/modeller/). ModBase currently contains 10 355 444 reliable models for domains in 2 421 920 unique protein sequences. ModBase allows users to update comparative models on demand, and request modeling of additional sequences through an interface to the ModWeb modeling server (http://salilab.org/modweb). ModBase models are available through the ModBase interface as well as the Protein Model Portal (http://www.proteinmodelportal.org/). Recently developed associated resources include the SALIGN server for multiple sequence and structure alignment (http://salilab.org/salign), the ModEval server for predicting the accuracy of protein structure models (http://salilab.org/modeval), the PCSS server for predicting which peptides bind to a given protein (http://salilab.org/pcss) and the FoXS server for calculating and fitting Small Angle X-ray Scattering profiles (http://salilab.org/foxs).
doi:10.1093/nar/gkq1091
PMCID: PMC3013688  PMID: 21097780
25.  Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes† 
Biochemistry  2009;48(21):4567-4576.
Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300) while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea (gi| 44371129). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of L-Xaa-L-Xaa dipeptides where the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were L-Ala-L-Leu with values of kcat and kcat/Km of 37 s−1 and 1.1 × 105 M−1 s−1, respectively, and N-formyl-L-Tyr with values of kcat and kcat/Km of 33 s−1 and 3.9 × 105 M−1 s−1, respectively. The best substrate identified for Sgx9355e was L-Ala-L-Phe will values of kcat and kcat/Km of 0.41 s−1 and 5.8 × 103 M−1 s−1. The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 Å with L-methionine bound in the active site. The α-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The α-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.
doi:10.1021/bi900453u
PMCID: PMC2748308  PMID: 19358546

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