Christopher J. Arthur

A Mammalian Type I Fatty Acid Synthase Acyl Carrier Protein Domain Does Not Sequester Acyl Chains

The synthases that produce fatty acids in mammals (FASs) are arranged as large multidomain polypeptides. The growing fatty acid chain is bound covalently during chain elongation and reduction to the acyl carrier protein (ACP) domain that is then able to access each catalytic site. In this work we report the high-resolution nuclear magnetic resonance (NMR) solution structure of the isolated rat fatty acid synthase apoACP domain. The final ensemble of NMR structures and backbone 15N relaxation studies show that apoACP adopts a single, well defined fold. On conversion to the holo form, several small chemical shift changes are observed on the ACP for residues surrounding the phosphopantetheine attachment site (as monitored by backbone 1H-15N correlation experiments). However, there are negligible chemical shift changes when the holo form is modified to either the hexanoyl or palmitoyl forms. For further NMR analysis, a 13C,15N-labeled hexanoyl-ACP sample was prepared and full chemical shift assignments completed. Analysis of two-dimensional F2-filtered and three-dimensional 13C-edited nuclear Overhauser effect spectroscopy experiments revealed no detectable NOEs to the acyl chain. These experiments demonstrate that unlike other FAS ACPs studied, this Type I ACP does not sequester a covalently linked acyl moiety, although transient interactions cannot be ruled out. This is an important mechanistic difference between the ACPs from Type I and Type II FASs and may be significant for the modulation and regulation of these important mega-synthases.

An ACP structural switch: conformational differences between the apo and holo forms of the actinorhodin polyketide synthase acyl carrier protein.

The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well‐ordered backbone atoms to the average coordinates of 0.37 and 0.42 Å, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo‐synthase (ACPS) assays indicated that apo‐ACP has a twofold higher affinity (Kd of 1.1 μM) than holo‐ACP (Kd of 2.1 μM) for ACPS. Site‐directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo‐ and holo‐ACP structures with known models of the Bacillus subtilis FAS ACP–holo‐acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo‐ and holo‐ACP could play a role in dissociation of the ACP–ACPS complex.

The type I rat fatty acid synthase ACP shows structural homology and analogous biochemical properties to type II ACPs

While X-ray and NMR structures are now available for most components of the Type II fatty acid synthase (FAS), there are no structures for Type I FAS domains. A region from the mammalian (rat) FAS, including the putative acyl carrier protein (ACP), has been cloned and over-expressed. Here we report multinuclear, multidimensional NMR studies which show that this isolated ACP domain contains four alpha-helices (residues 8-16 [1]; 41-51 [2]; 58-63 [3] and 66-74 [4]) and an overall global fold characteristic of ACPs from both Type II FAS and polyketide synthases (PKSs). The overall length of the structured ACP domain (67 residues) is smaller than the structured regions of the Eschericia coli FAS ACP (75 residues), the actinorhodin PKS ACP (78 residues) and the Bacillus subtilis FAS ACP (76 residues). We further show that the rat FAS ACP is recognised as an efficient substrate by enzymes known to modify Type II ACPs including phosphopantetheinyl and malonyl transferases, but not by the heterologous S. coelicolor minimal polyketide synthase.

Catalytic relationships between type I and type II iterative polyketide synthases: The Aspergillus parasiticus norsolorinic acid synthase.

Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus Aspergillus parasiticus. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and β‐ketoacyl‐ACP synthase (KS) catalytic domains. Expression of these gene fragments in Escherichia coli led to the production of soluble ACP and AT proteins. Coexpression of ACP with E. coli holo‐ACP synthase (ACPS) let to production of NSAS holo‐ACP, which could also be formed in vitro by using Streptomyces coelicolor ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self‐malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS holo‐ACP serves as substrate for S. coelicolor MCAT, S. coelicolor actinorhodin holo‐ACP and NSAS AT domain‐catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS holo‐ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS holo‐ACP. The NSAS holo‐ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.

Recognition of Intermediate Functionality by Acyl Carrier Protein over a Complete Cycle of Fatty Acid Biosynthesis

It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis.

Structure and malonyl CoA-ACP transacylase binding of streptomyces coelicolor fatty acid synthase acyl carrier protein.

Malonylation of an acyl carrier protein (ACP) by malonyl Coenzyme A-ACP transacylase (MCAT) is fundamental to bacterial fatty acid biosynthesis. Here, we report the structure of the Steptomyces coelicolor (Sc) fatty acid synthase (FAS) ACP and studies of its binding to MCAT. The carrier protein adopts an alpha-helical bundle structure common to other known carrier proteins. The Sc FAS ACP shows close structural homology with other fatty acid ACPs and less similarity with Sc actinorhodin (act) polyketide synthase (PKS) ACP where the orientation of helix I differs. NMR experiments were used to map the binding of ACP to MCAT. This data suggests that Sc FAS ACP interacts with MCAT through the negatively charged helix II of ACP, consistent with proposed models for ACP recognition by other FAS enzymes. Differential roles for residues at the interface are demonstrated using site-directed mutagenesis and in vitro assays. MCAT has been suggested, moreover, to participate in bacterial polyketide synthesis in vivo. We demonstrate that the affinity of the polyketide synthase ACP for MCAT is lower than that of the FAS ACP. Mutagenesis of homologous helix II residues on the polyketide synthase ACP suggests that the PKS ACP may bind to MCAT in a different manner than the FAS counterpart.

Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus

Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty‐four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides salivary glands, as well as several members of the D7 family and protease inhibitors with putative anticoagulant activity. In addition, several families of abundant proteins with unknown function were identified including some of the major candidate allergens that cause insect bite hypersensitivity in horses.

Detection of humoral response using a recombinant heat shock protein 70 (dnaK) of Mycoplasma haemofelis in experimentally and naturally hemoplasma infected-cats

ABSTRACT Hemoplasmas is the trivial name given to a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. Of the feline hemoplasmas, Mycoplasma haemofelis is the most pathogenic, while “Candidatus Mycoplasma haemominutum” and “Candidatus Mycoplasma turicensis” are less pathogenic. Shotgun libraries of fragmented M. haemofelis genomic DNA were constructed, and random colonies were selected for DNA sequencing. In silico-translated amino acid sequences of putative open reading frames were compared to mass spectrometry data from M. haemofelis protein spots identified as being immunogenic by two-dimensional gel electrophoresis and Western blotting. Three of the spots matched the predicted sequences of a heat shock protein 70 (DnaK) homolog, elongation factor Ts, and a fragment of phosphoglycerate kinase found during library screening. A full-length copy of the M. haemofelis dnaK gene was cloned into Escherichia coli and recombinantly expressed. Recombinant M. haemofelis DnaK was purified and then used in Western blotting and an enzyme-linked immunosorbent assay (ELISA) to investigate the humoral immune response during acute infection in cats experimentally infected with M. haemofelis, “Ca. Mycoplasma haemominutum,” or “Ca. Mycoplasma turicensis”. The recombinant M. haemofelis DnaK ELISA also was used to screen clinical samples submitted for hemoplasma PCR testing to a commercial laboratory (n = 254). Experimentally infected cats became seropositive following infection, with a greater and earlier antibody response seen in cats inoculated with M. haemofelis than those seen in cats inoculated with “Ca. Mycoplasma haemominutum” or “Ca. Mycoplasma turicensis,” by both Western blotting and ELISA. Of the clinical samples, 31.1% had antibodies detected by the ELISA but only 9.8% were positive by PCR for one or more hemoplasmas.

Synthesis and characterisation of acyl carrier protein bound polyketide analogues.

Polyketide synthases (PKSs) are widespread in plants, bacteria and fungi. They are responsible for the biosynthesis of an enormous range of organic compounds, many of which have important pharmaceutical and agrochemical properties. Three types of synthases have been documented. Type I systems consist of very large multifunctional proteins which can be either processive (for example the modular systems responsible for macrolide synthesis) or iterative (for example the lovastatin nonaketide synthase from Aspergillus terreus ). The iterative Type II PKS systems consist of complexes of monofunctional proteins exemplified by the actinorhodin (act) 1 PKS from Streptomyces coelicolor. Type III systems are responsible for the synthesis of chalcones and stilbenes in plants and polyhydroxy phenols in bacteria. All PKS possess the key -ketoacyl synthase domain responsible for the C C bond forming reaction. In Type I and Type II systems the growing acyl chain is covalently attached to the terminal thiol of a phosphopantetheine (PP) prosthetic group on an acyl carrier protein (ACP). In the act PKS a minimal set of proteins has been identified which is capable of synthesising polyketides in vitro from malonyl CoA. These proteins are KS , responsible for C C bond formation, KS responsible inter alia for starter unit production and the ACP (Figure 1). We have extensively studied these components and their biochemical activities in vitro. In short, these proteins load malonyl units onto the terminal PP thiol of the ACP (Scheme 1). KS then produces acetyl-ACP and KS performs seven decarboxylative condensations to produce a putative ACP-bound octaketide. The subsequent cyclisation and release of this octaketide, probably controlled by the minimal PKS, yields SEK4 (2) and SEK4b (3) as the products. We studied the acyl ACP intermediates formed during PKS catalysis. Novel acyl ACPs could act as surrogate intermediates or starter units for PKS and may lead to the synthesis of novel compounds in vitro. Acyl ACPs could also be substrates for other concerning bonding interactions that involve sulfur, Dr. M. T. Stubbs for his help in the crystal structure determinations, and Prof. G. A. Garcia for providing samples of tRNA.

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis

Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes.Analysis showed that in hemoplasmas genes involved in carbohydrate metabolism are limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source. The majority of the pentose phosphate pathway enzymes that catalyze the de novo synthesis of ribonucleotides were absent, as were cell division protein FtsZ and chaperonins GroEL/ES. Uncharacterized protein paralogs containing putative surface expression motifs, comprised 62% of M. haemofelis and 19% of Mycoplasma suis genome coverage respectively, the majority of which were present in a small number of unstructured islands. Limited mass spectrometry and immunoblot data matched a number of characterized proteins and uncharacterized paralogs, confirming their expression and immunogenicity in vivo.These data have allowed further characterization of these important pathogens, including their limited metabolic capabilities, which may contribute to their uncultivatable status. A number of immunogenic proteins, and a potential mechanism for host immune system evasion, have been identified.