Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.

SUMMARY FoxO transcription factors regulate the transcription of genes that control metabolism, cellular proliferation, stress tolerance and possibly lifespan. A number of post-translational modifications within the forkhead DNA binding domain regulate FoxO mediated transcription. We report the crystal structures of FoxO1 bound to three different DNA elements and measure the change in FoxO1-DNA affinity with acetylation and phosphorylation. The structures reveal additional contacts and increased DNA distortion for the highest affinity DNA site. The flexible wing 2 region of the forkhead domain was not observed in the structures but is necessary for DNA-binding, and we show that p300 acetylation in wing 2 reduces DNA affinity. We also show that MST1 phosphorylation of FoxO1 prevents high affinity DNA binding. The observation that FoxO-DNA affinity varies between response elements and with post-translational modifications suggests that modulation of FoxO-DNA affinity is an important component of FoxO regulation in health and misregulation in disease.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. In eukaryotes and Gram-negative bacteria, FGAR-AT is encoded by the purL gene as a multidomain protein with a molecular mass of about 140 kDa. In Gram-positive bacteria and archaebacteria FGAR-AT is a complex of three proteins: PurS, PurL, and PurQ. We have determined the structure of FGAR-AT (PurL) from Salmonella typhimurium at 1.9 A resolution using X-ray crystallography. PurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined. The structure reveals four domains: an N-terminal domain structurally homologous to a PurS dimer, a linker region, an FGAM synthetase domain homologous to an aminoimidazole ribonucleotide synthetase (PurM) dimer, and a triad glutaminase domain. The domains are intricately linked by interdomain interactions and peptide connectors. The fold common to PurM and the central region of PurL represents a superfamily for which HypE, SelD, and ThiL are predicted to be members. A structural ADP molecule was found bound to a site related to the putative active site by pseudo-2-fold symmetry and two sulfate ions were found at the putative active site. These observations and the structural similarities between PurM and StPurL were used to model the substrates FGAR and ATP in the StPurL active site. A glutamylthioester intermediate was found in the glutaminase domain at Cys1135. The N-terminal (PurS-like) domain is hypothesized to form the putative channel through which ammonia passes from the glutaminase domain to the FGAM synthetase domain.

The discovery of histone-demethylating enzymes has revealed yet another reversible histone modification mark. In this review, we describe the structural and chemical insights that we have now derived underlying the activity of these enzymes. The recent cocrystal structures of LSD1 bound to a proparylamine-derivatized histone H3 peptide and JHDM structures bound to two different methylated histone H3 peptides illustrate the steric requirements and structural basis for substrate specificity.Histones are evolutionarily conserved proteins that are the building blocks of the nucleoprotein chromatin structure that packages DNA within the eukaryotic nucleus. Chromatin contains individual nucleosomal core particles with eight core histone proteins, two copies each of histones H3, H4, H2B, and H2A, with 146 bp of DNA wrapped around it (1, 2). The histone proteins each contain a globular core that is surrounded by DNA within the nucleosome and N-terminal tails that protrude out of the core/DNA region and are subject to a diverse array of post-translational modifications, including acetylation, methylation, SUMOylation, and ubiquitination. The combinatorial effect of these post-translational modifications affects key DNA regulatory processes such as DNA replication, DNA repair, and transcriptional activation and repression.Most if not all of the histone modifications are dynamic in nature, providing reversible modes of regulation. However, until very recently, histone methylation was thought to be a stable genomic imprint. This dogma was attributed, in part, to the thermodynamic stability of the N-CH 3 bond and supporting biochemical studies demonstrating comparable turnover rates of bulk histones and the methyl groups on histone lysine and arginine residues in mammalian cells (3). The major methylation sites within histone tails are the basic amino acid side chains of lysine and arginine residues. Lysines within histones can be mono-, di-, or trimethylated on the ⑀-nitrogen, and arginines are mono-or dimethylated on the guanidinium group (4 -6). Lysine-specific methylation is catalyzed using a highly conserved class of enzymes, histone methyltransferases. Histone methyltransferases utilize S-adenosyl-L-methionine as the methyl group donor (7,8). Early studies using metabolic labeling followed by sequencing of bulk histones have shown that several lysine residues, including lysines 4, 9, 27, and 36 of histone H3 and lysine 20 of histone H4, are preferred sites of methylation (9).Histone arginine methylation is generally linked to transcriptional activation, whereas histone lysine methylation can correlate with either transcriptional activation or repression, depending on the site and status of methylation (10, 11). Experiments have shown that methylation at lysines 4 (H3K4), 36 (H3K36), and 79 (H3K79) of histone H3 leads to activation of euchromatic genes, whereas methylation at lysines 9 (H3K9) and 27 (H3K27) of histone H3 and methylation at lysine 20 of histone H4 (H4K20) are marks of repressed chromatin (10, 12). This w...

Phenol and its derivatives are common pollutants that are present in industrial discharge and are major xenobiotics that lead to water pollution. To monitor as well as improve water quality, attempts have been made in the past to engineer bacterial in vivo biosensors. However, due to the paucity of structural information, there is insufficiency in gauging the factors that lead to high sensitivity and selectivity, thereby impeding development. Here, we present the crystal structure of the sensor domain of MopR (MopR(AB)) from Acinetobacter calcoaceticus in complex with phenol and its derivatives to a maximum resolution of 2.5 Å. The structure reveals that the N-terminal residues 21-47 possess a unique fold, which are involved in stabilization of the biological dimer, and the central ligand binding domain belongs to the "nitric oxide signaling and golgi transport" fold, commonly present in eukaryotic proteins that bind long-chain fatty acids. In addition, MopR(AB) nests a zinc atom within a novel zinc binding motif, crucial for maintaining structural integrity. We propose that this motif is crucial for orchestrated motions associated with the formation of the effector binding pocket. Our studies reveal that residues W134 and H106 play an important role in ligand binding and are the key selectivity determinants. Furthermore, comparative analysis of MopR with XylR and DmpR sensor domains enabled the design of a MopR binding pocket that is competent in binding DmpR-specific ligands. Collectively, these findings pave way towards development of specific/broad based biosensors, which can act as useful tools for detection of this class of pollutants.

The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.

NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 10(5) M(-1) s(-1)), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 Å. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. Small PurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the apo form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analog, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. Availability of a ternary complex enabled mapping of the active site thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. A surprising discovery, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculations about the regulatory role of the auxiliary site in PurLSQ complex formation as well as the evolutionary relationship of PurL's from different organisms.The purine biosynthetic pathway is ubiquitous in most living organisms, and it is a ten-step process for converting phosphoribosyl pyrophosphate to inosine monophosphate. Formyl glycinamide ribonucleotide amidotransferase (FGAR-AT), also known by its gene product † This work was supported by the NIH grants RR15301 and GM073220 (SEE). SEE is indebted to the W. M. Keck Foundation and the Lucille P. Markey Charitable Trust. AAH was supported by a NSF predoctoral fellowship. AAH and JS were supported by NIH Grant GM32191. The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems at MIT is supported by grants (NSF-0070319 and NIH GM68762). Supporting Information Available A table of primers used in TmPurL cloning and mutagenesis, a table summarizing the SV-AUC results, the Coomassie-stained gels for StPurL and mutants, a graph of the distribution of species obtained after SEDFIT analysis of SV-AUC data for wild type and mutant TmPurL, a topology diagram of TmPurL, circular dichroism spectra for wild type and mutant TmPurL, and an elution profile of wild type TmPurL after incubation with radioactive ATP. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript name, PurL, catalyzes the fourth...