Metal fluorides-multi-functional tools for the study of phosphoryl transfer enzymes, a practical guide.

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate.

Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase.

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate.

Photocobilins integrate B and bilin photochemistry for enzyme control.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B) can act as a light-sensing chromophore heralded a new field of B-photobiology. Although microbial genome analysis indicates that photoactive B-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking.

Backbone H, C and N resonance assignment of the ubiquitin specific protease 7 catalytic domain (residues 208-554) in complex with a small molecule ligand.

The backbone H, C and N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide H-N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed H, C, N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.

A Chiral F NMR Reporter of Foldamer Conformation in Bilayers.

Understanding and controlling peptide foldamer conformation in phospholipid bilayers is a key step toward their use as molecular information relays in membranes. To this end, a new F "reporter" tag has been developed and attached to dynamic peptide foldamers. The ()-1-(trifluoromethyl)ethylamido (()-TFEA) reporter was attached to the C-terminus of α-amino--butyric acid (Aib) foldamers.

An Enzyme with High Catalytic Proficiency Utilizes Distal Site Substrate Binding Energy to Stabilize the Closed State but at the Expense of Substrate Inhibition.

Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state.

Microarray screening reveals two non-conventional SUMO-binding modules linked to DNA repair by non-homologous end-joining.

SUMOylation is critical for numerous cellular signalling pathways, including the maintenance of genome integrity via the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Using systematic human proteome microarray screening combined with widely applicable carbene footprinting, genetic code expansion and high-resolution structural profiling, we define two non-conventional and topology-selective SUMO2-binding regions on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ).

Spinning sugars in antigen biosynthesis: characterization of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases.

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii.

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase.

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied.

H, C, N backbone resonance assignment for the 1-164 construct of human XRCC4.

DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as-if mis- or unrepaired-they can cause cell death or lead to genome instability, which in turn can cause cancer. DSBs are repaired by two major pathways termed homologous recombination and non-homologous end-joining (NHEJ). NHEJ is responsible for repairing the vast majority of DSBs arising in human cells.

Structure and Mechanism of PA0254/HudA, a prFMN-Dependent Pyrrole-2-carboxylic Acid Decarboxylase Linked to Virulence.

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor.

Allomorphy as a mechanism of post-translational control of enzyme activity.

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate.

Structural effects of the highly protective V127 polymorphism on human prion protein.

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding.

Mapping Hidden Residual Structure within the Myc bHLH-LZ Domain Using Chemical Denaturant Titration.

Intrinsically disordered proteins (IDPs) underpin biological regulation and hence are highly desirable drug-development targets. NMR is normally the tool of choice for studying the conformational preferences of IDPs, but the association of regions with residual structure into partially collapsed states can lead to poor spectral quality. The bHLH-LZ domain of the oncoprotein Myc is an archetypal example of such behavior.

Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.

Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy).

Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol--methyltransferase.

Catechol--methyltransferase (COMT) is a model S-adenosyl-l-methionine (SAM) dependent methyl transferase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals. Despite extensive study, there is no consensus view of the physical basis of catalysis in COMT. Further progress requires experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate.

Isotopically labeled flavoenzymes and their uses in probing reaction mechanisms.

The incorporation of stable isotopes into proteins is beneficial or essential for a range of experiments, including NMR, neutron scattering and reflectometry, proteomic mass spectrometry, vibrational spectroscopy and "heavy" enzyme kinetic isotope effect (KIE) measurements. Here, we present detailed protocols for the stable isotopic labeling of pentaerythritol tetranitrate reductase (PETNR) via recombinant expression in E. coli.

Enzymatic Carboxylation of 2-Furoic Acid Yields 2,5-Furandicarboxylic Acid (FDCA).

The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO levels in vitro.

Myc phosphorylation in its basic helix-loop-helix region destabilizes transient α-helical structures, disrupting Max and DNA binding.

Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity.

The role of conserved residues in Fdc decarboxylase in prenylated flavin mononucleotide oxidative maturation, cofactor isomerization, and catalysis.

The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu-Arg-Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMN and prFMN, have been observed. It also has been suggested that only the prFMN form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor.

H, N and C backbone resonance assignments of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step.

H, N, C backbone resonance assignments of human phosphoglycerate kinase in a transition state analogue complex with ADP, 3-phosphoglycerate and magnesium trifluoride.

Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes.

Assessing the Influence of Mutation on GTPase Transition States by Using X-ray Crystallography, F NMR, and DFT Approaches.

We report X-ray crystallographic and F NMR studies of the G-protein RhoA complexed with MgF , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain.

H, N, C backbone resonance assignments of human soluble catechol O-methyltransferase in complex with S-adenosyl-L-methionine and 3,5-dinitrocatechol.

Catechol O-methyltransferase (COMT) is an enzyme that plays a major role in catechol neurotransmitter deactivation. Inhibition of COMT can increase neurotransmitter levels, which provides a means of treatment for Parkinson's disease, schizophrenia and depression. COMT exists as two isozymes: a soluble cytoplasmic form (S-COMT), expressed in the liver and kidneys and a membrane-bound form (MB-COMT), found mostly in the brain.