Molecular basis for control of antibiotic production by a bacterial hormone.

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.

Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B.

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of ß-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on ß-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of ß-lactam acylation.

Binding of Distinct Substrate Conformations Enables Hydroxylation of Remote Sites in Thaxtomin D by Cytochrome P450 TxtC.

Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation, and natural product biosynthesis. Here we report biochemical, structural, and theoretical studies of TxtC, an unusual bifunctional CYP involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the α-carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the π-face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally distinct binding mode in which the α-hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the α-hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohydroxylated intermediate.

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds.

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a ß-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS.

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity.

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

Ferric ion (hydr)oxo clusters in the "Venus flytrap" cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies.

Isothermal calorimetric studies of the binding of iron(III) citrate to ferric ion binding protein from Neisseria gonorrhoeae suggested the complexation of a tetranuclear iron(III) cluster as a single step binding event (apparent binding constant K(app) (ITC) = 6.0(5) × 10(5) M(-1)). High-resolution Fourier transform ion cyclotron resonance mass spectrometric data supported the binding of a tetranuclear oxo(hydroxo) iron(III) cluster of formula [Fe(4)O(2)(OH)(4)(H(2)O)(cit)](+) in the interdomain binding cleft of FbpA. The mutant H9Y-nFbpA showed a twofold increase in the apparent binding constant [K(app) (ITC) = 1.1(7) × 10(6) M(-1)] for the tetranuclear iron(III) cluster compared to the wild-type protein. Mössbauer spectra of Escherichia coli cells overexpressing FbpA and cultured in the presence of added (57)Fe citrate were indicative of the presence of dinuclear and polynuclear clusters. FbpA therefore appears to have a strong affinity for iron clusters in iron-rich environments, a property which might endow the protein with new biological functions.

Arabidopsis thaliana circadian clock is regulated by the small GTPase LIP1.

BACKGROUND: At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative-feedback loops and generate a basic approximately 24 hr oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. RESULTS: The lip1-1 (light insensitive period 1) mutant isolated from the model plant Arabidopsis thaliana displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. In wild-type plants, period length shortens with increasing light fluence rates and the phase of rhythms can be shifted by light pulses administered to dark-adapted plants. In contrast, in lip1-1, period length is nearly insensitive to light intensity and significantly larger phase shifts (delays) can be induced during the subjective night. The mutant also displays elevated photomorphogenic responses to red and blue light, which cannot be explained by the circadian defect, suggesting distinct functions for LIP1 in the circadian light input and photomorphogenesis. The LIP1 gene encodes a functional, plant-specific atypical small GTPase, and therefore we postulate that it acts similarly to ZEITLUPE at postranscriptional level. CONCLUSIONS: LIP1 represents the first small GTPase implicated in the circadian system of plants. LIP1 plays a unique negative role in controlling circadian light input and is required for precise entrainment of the plant clock.

Inhibition of prolyl oligopeptidase with a synthetic unnatural dipeptide.

A new inhibitor, containing a linked proline-piperidine structure, for the enzyme prolyl oligopeptidase (POP) has been synthesised and demonstrated to bind covalently with the enzyme at the active site. This provides evidence that covalent inhibitors of POP do not have to be limited to structures containing five-membered N-containing heterocyclic rings.

Crystallization and preliminary X-ray analysis of a D-alanyl-D-alanine ligase (EcDdlB) from Escherichia coli.

A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and D-alanyl-D-alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and D-alanyl-D-alanine, representing the product-inhibited complex. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.0, b = 97.6, c = 109.5 A and a = 51.2, b = 97.8, c = 110.1 A, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4 A resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation.

Structural basis for chain release from the enacyloxin polyketide synthase.

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Crystal structure and functional assignment of YfaU, a metal ion dependent class II aldolase from Escherichia coli K12.

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of the Escherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg (2+)-pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the homologous 2-dehydro-3-deoxygalactarate aldolase (DDGA). Enzyme kinetic measurements show activity with the probable physiological substrate 2-keto-3-deoxy- l-rhamnonate, supporting the functional assignment, as well as the structurally similar 2-keto-3-deoxy- l-mannonate and 2-keto-3-deoxy- l-lyxonate (see accompanying paper: Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2008) Biochemistry 47, 9944-9954). YfaU has similar activity toward the HpcH substrate 4-hydroxy-2-ketoheptane-1,7-dioate and synthetic substrates 4-hydroxy-2-ketopentanoic acid and 4-hydroxy-2-ketohexanoic acid. This indicates a relaxed substrate specificity that complicates the functional assignment of members of this enzyme superfamily. Crystal structures suggest these enzymes use an Asp-His intersubunit dyad to activate a metal-bound water or hydroxide for proton transfer during catalysis.

Structure and mechanism of HpcH: a metal ion dependent class II aldolase from the homoprotocatechuate degradation pathway of Escherichia coli.

Microorganisms are adept at degrading chemically resistant aromatic compounds. One of the longest and most well characterized aromatic catabolic pathways is the 4-hydroxyphenylacetic acid degradation pathway of Escherichia coli. The final step involves the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate into pyruvate and succinic semialdehyde. This reaction is catalyzed by 4-hydroxy-2-oxo-heptane-1,7-dioate aldolase (HpcH), a member of the divalent metal ion dependent class II aldolase enzymes that have great biosynthetic potential. We have solved the crystal structure of HpcH in the apo form, and with magnesium and the substrate analogue oxamate bound, to 1.6 A and 2.0 A, respectively. Comparison with similar structures of the homologous 2-dehydro-3-deoxygalactarate aldolase, coupled with site-directed mutagenesis data, implicate histidine 45 and arginine 70 as key catalytic residues.

Structure and mechanism of HpcG, a hydratase in the homoprotocatechuate degradation pathway of Escherichia coli.

HpcG catalyses the hydration of a carbon-carbon double bond without the aid of any cofactor other than a simple divalent metal ion such as Mg(2+). Since the substrate has a nearby carbonyl group, it is believed that it first isomerises to form a pair of conjugated double bonds in the enol tautomer before Michael addition of water. Previous chemical studies of the reaction, and that of the related enzyme MhpD, have failed to provide a clear picture of the mechanism. The substrate itself is unstable, preventing co-crystallisation or soaking of crystals, but oxalate is a strong competitive inhibitor. We have solved the crystal structure of the protein in the apo form, and with magnesium and oxalate bound. Modelling substrate into the active site suggests the attacking water molecule is not part of the metal coordination shell, in contrast to a previous proposal. Our model suggests that geometrically strained cis isomer intermediates do not lie on the reaction pathway, and that separate groups are involved in the isomerisation and hydration steps.

Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling.

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.

Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine.

D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.

Structure-function properties of prolyl oligopeptidase family enzymes.

Prolyl oligopeptidase family enzymes regulate the activity of biologically active peptides and peptide hormones, and they are implicated in diseases, including amnesia, depression, diabetes, and trypanosomiasis. Distinctively, these enzymes hydrolyze only relatively short peptide substrates, while large structured peptides and proteins are not usually cleaved. Prolyl oligopeptidase has a C-terminal alpha/beta-hydrolase catalytic domain that is similar to lipases and esterases. An N-terminal beta-propeller domain regulates access to the buried active site, explaining the observed oligopeptidase activity. The catalytic and regulatory mechanisms have been investigated using a combination of X-ray crystallography, site-directed mutagenesis, and enzyme kinetic measurements. Crystal structures have now been determined for representative members of three of the four subfamilies and are facilitating a better understanding of the structure-function properties of these physiologically and pharmaceutically important enzymes.

Expression, purification and preliminary crystallographic analysis of oligopeptidase B from Trypanosoma brucei.

African sleeping sickness, also called trypanosomiasis, is a significant cause of morbidity and mortality in sub-Saharan Africa. Peptidases from Trypanosoma brucei, the causative agent, include the serine peptidase oligopeptidase B, a documented virulence factor and therapeutic target. Determination of the three-dimensional structure of oligopeptidase B is desirable to facilitate the development of novel inhibitors. Oligopeptidase B was overexpressed in Escherichia coli as an N-terminally hexahistidine-tagged fusion protein, purified using metal-affinity chromatography and crystallized using the hanging-drop vapour-diffusion technique in 7%(w/v) polyethylene glycol 6000, 1 M LiCl, 0.1 M bis-tris propane pH 7.5. Diffraction data to 2.7 angstroms resolution were collected using synchrotron radiation. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 124.5, c = 249.9 angstroms. A complete data set to 2.7 angstroms was collected using synchrotron radiation.

Expression, purification and crystallization of 2-oxo-hept-4-ene-1,7-dioate hydratase (HpcG) from Escherichia coli C.

The gene encoding 2-oxo-hept-3-ene-1,7-dioic acid (OHED) hydratase (HpcG) was cloned into the high-expression plasmid pET26b and overexpressed in Escherichia coli BL21(DE3). The enzyme was purified in three steps to greater than 95% purity prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K in a number of screening conditions. Crystals measuring up to 1.5 mm in their longest dimension were grown from solutions containing polyethylene glycol 20 000. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 136, b = 136, c = 192 A. A complete data set was collected to 2.1 A from a single cryocooled crystal at 100 K using synchrotron radiation.

Concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding.

Prolyl oligopeptidase contains a peptidase domain and its catalytic triad is covered by the central tunnel of a seven-bladed beta-propeller. This domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. The apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. Two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart, or the peptidase and/or propeller domains move to create an entry site at the domain interface. Engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. This indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action.

Expression, purification and preliminary crystallographic analysis of 2,4-dihydroxy-hepta-2-ene-1,7-dioate aldolase (HpcH) from Escherichia coli C.

The gene encoding 2,4-dihydroxy-hepta-2-ene-1,7-dioate (HHED) aldolase (HpcH; EC 4.1.2) from Escherichia coli C was cloned into the high-expression plasmid pProEx-HTa and overexpressed in E. coli BL21 (DE3). The 28 kDa enzyme was purified using immobilized metal-affinity and size-exclusion chromatography prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K from a number of screening conditions. Type I crystals grown in a solution containing 0.4 M ammonium dihydrogen phosphate belong to space group R32, with unit-cell parameters a = b = 128.92, c = 175.30 A. Type II crystals grown in a solution containing 0.5 M sodium chloride, 0.1 M sodium citrate pH 5.5 belong to space group I222, with unit-cell parameters a = 133.39, b = 155.39, c = 168.80 A. Complete data sets were collected to 1.6 and 2.0 A from type I and type II crystals, respectively, using synchrotron radiation.