A Pseudoisostructural Type II DAH7PS Enzyme from Pseudomonas aeruginosa: Alternative Evolutionary Strategies to Control Shikimate Pathway Flux.

The shikimate pathway is responsible for the biosynthesis of key aromatic metabolites in microorganisms and plants. The enzyme 3-deoxy-d- arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the pathway and DAH7PSs are classified as either type I or type II. The DAH7PSs from Pseudomonas aeruginosa are of particular interest as open reading frames encoding four putative DAH7PS isoenzymes, two classified as type Iα and two classified as type II, have been identified. Here, the structure of a type II DAH7PS enzyme from P. aeruginosa (PAO1) has been determined at 1.54 Å resolution, in complex with its allosteric inhibitor tryptophan. Structural differences in the extra-barrel elements, when compared to other type II DAH7PS enzymes, directly relate to the formation of a distinct quaternary conformation with consequences for allosteric function and the control of flux to branching pathways. In contrast to the well-characterized Mycobacterium tuberculosis type II DAH7PS, which binds multiple allosteric inhibitors, this PaeDAH7PSPA2843 is observed to be modestly allosterically inhibited by a single aromatic amino acid, tryptophan. In addition, structures in complex with tyrosine or with no allosteric ligand bound were determined. These structures provide new insights into the linkages between the active and allosteric sites. With four putative DAH7PS enzymes, P. aeruginosa appears to have evolved control of shikimate pathway flux at the genetic level, rather than control by multiple allosteric effectors to a single type II DAH7PS, as in M. tuberculosis. Type II DAH7PSs, thus, appear to have a more varied evolutionary trajectory than previously indicated.

Heterologous Biosynthesis of Nodulisporic Acid F.

Nodulisporic acids comprise a group of valuable indole diterpenes that exhibit potent insecticidal activities. We report the identification of a gene cluster in the genome of the filamentous fungus Hypoxylon pulicicidum (Nodulisporium sp.) that contains genes responsible for the biosynthesis of nodulisporic acids. Using Penicillium paxilli as a heterologous host, and through pathway reconstitution experiments, we identified the function of four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F (NAF). Two of these genes (nodM and nodW) are especially significant as they encode enzymes with previously unreported functionality: nodM encodes a 3-geranylgeranylindole epoxidase capable of catalyzing only a single epoxidation step to prime formation of the distinctive ring structure of nodulisporic acids, and nodW encodes the first reported gene product capable of introducing a carboxylic acid moiety to an indole diterpene core structure that acts as a reactive handle for further modification. Here, we present the enzymatic basis for the biosynthetic branch point that gives rise to nodulisporic acids.

Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation.

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.

Conformational Dynamics and Allostery in Pyruvate Kinase.

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ß-strands within the (ß/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site for N-acetylneuraminic acid synthase.

N-Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesized in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalyzed by a domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). NANAS comprises two distinct domains; an N-terminal catalytic (ß/α)8 barrel linked to a C-terminal antifreeze protein-like (AFPL) domain. We have investigated the role of the AFPL domain by characterizing a truncated variant of NmeNANAS, which was discovered to be soluble yet inactive. Analytical ultracentrifugation and analytical size exclusion were used to probe the quaternary state of the NmeNANAS truncation, and revealed that loss of the AFPL domain destabilizes the dimeric form of the enzyme. The results from this study thereby demonstrate that the AFPL domain plays a critical role for both the catalytic function and quaternary structure stability of NANAS. Small angle X-ray scattering, molecular dynamics simulations, and amino acid substitutions expose a complex hydrogen-bonding relay, which links the roles of the catalytic and AFPL domains across subunit boundaries.

Biological and biochemical characterization of HIV-1 Gag/dgp41 virus-like particles expressed in Nicotiana benthamiana.

The transmembrane HIV-1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4⁺ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus-based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus.

The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Šresolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.

Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley.

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3'-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends > or = 1230 bp from the 3' end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 --> 3),(1 --> 4)-beta-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-(14)C-Glc into cellulose and into mixed-linkage (1 --> 3),(1 --> 4)-beta-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 --> 3),(1 --> 4)-beta-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis.

Nodulisporic acid E biosynthesis: in vivo characterisation of NodD1, an indole-diterpene prenyltransferase that acts on an emindole SB derived indole-diterpene scaffold.

Prenylation of aromatic compounds is a key tailoring reaction in biosynthesis of bioactive indole-diterpenes. Here, we identify NodD1 as the enzyme responsible for the bisprenylation of nodulisporic acid F. This prenyltransferase showed a preference for its natural indole-diterpene substrate whereas other related enzymes were not able to catalyse this conversion.

Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.

In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity.

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

Grappling with anisotropic data, pseudo-merohedral twinning and pseudo-translational noncrystallographic symmetry: a case study involving pyruvate kinase.

Pyruvate kinase is a key regulatory enzyme involved in the glycolytic pathway. The crystal structure of Escherichia coli type I pyruvate kinase was first solved in 1995 at 2.5 Šresolution. However, the space group was ambiguous, being either primitive orthorhombic (P2(1)2(1)2(1)) or C-centred orthorhombic (C222(1)). Here, the structure determination and refinement of E. coli type I pyruvate kinase to 2.28 Šresolution are presented. Using the same crystallization conditions as reported previously, the enzyme was found to crystallize in space group P2(1). Determination of the space group was complicated owing to anisotropic data, pseudo-translational noncrystallographic symmetry and the pseudo-merohedrally twinned nature of the crystal, which was found to have very close to 50% twinning, leading to apparent orthorhombic symmetry and absences that were not inconsistent with P2(1)2(1)2(1). The unit cell contained two tetramers in the asymmetric unit (3720 residues) and, when compared with the orthorhombic structure, virtually all of the residues could be easily modelled into the density. Averaging of reflections into the lower symmetry space group with twinning provided tidier electron density that allowed ∼30 missing residues of the lid domain to be modelled for the first time. Moreover, residues in a flexible loop could be modelled and sulfate molecules are found in the allosteric binding domain, identifying the pocket that binds the allosteric activator fructose 1,6-bisphosphate in this isozyme for the first time. Lastly, we note the pedagogical benefits of difficult structures to emerging crystallographers.

Immunological Characterization of Plant-Based HIV-1 Gag/Dgp41 Virus-Like Particles.

It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR--a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine kinase from methicillin-resistant Staphylococcus aureus.

N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such as Escherichia coli, Staphylococcus aureus, Vibrio cholerae and V. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the `Nan-Nag cluster', which have proven to be essential for S. aureus growth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzyme N-acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine-5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine-6-phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed in E. coli BL21 (DE3) cells and purified via anion-exchange chromatography, hydrophobic interaction chromatography and size-exclusion chromatography. Purified N-acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space group P2. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide insights necessary for the development of future antimicrobials.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylmannosamine-6-phosphate 2-epimerase from methicillin-resistant Staphylococcus aureus.

Sialic acids are one of the most important carbohydrate classes in biology. Some bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This sequestration and subsequent catabolism of sialic acid require a cluster of genes known as the `Nan-Nag' cluster. The enzymes coded by these genes are important for pathogen colonization and persistence. Importantly, the Nan-Nag genes have proven to be essential for Staphylococcus aureus growth on sialic acids, suggesting that the pathway is a viable antibiotic drug target. The enzyme N-acetylmannosamine-6-phosphate 2-epimerase is involved in the catabolism of sialic acid; specifically, the enzyme converts N-acetylmannosamine-6-phosphate into N-acetylglucosamine-6-phosphate. The gene was cloned into an appropriate expression vector, and recombinant protein was expressed in Escherichia coli BL21 (DE3) cells and purified via a three-step procedure. Purified N-acetylmannosamine-6-phosphate 2-epimerase was screened for crystallization. The best crystal diffracted to a resolution of beyond 1.84 Å in space group P21212. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide us with the insights necessary for the development of future antibiotics.

Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41.

The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼ 1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.

Ultra-high resolution crystal structure of recombinant caprine ß-lactoglobulin.

ß-Lactoglobulin (ßlg) is the most abundant whey protein in the milks of ruminant animals. While bovine ßlg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine ßlg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine ßlg is dimeric (K(D)<5 µM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow's and goat's milk.