Discovery and Characterization of the Fully Decorated Nocardiosis-Associated Polyketide Natural Product.

The genomes of 40 strains of Nocardia, most of which were associated with life-threatening human infections, encode a highly conserved assembly line polyketide synthase designated as the NOCAP (NOCardiosis-Associated Polyketide) synthase, whose product structure has been previously described. Here we report the structure and inferred biosynthetic pathway of the fully decorated glycolipid natural product. Its structure reveals a fully substituted benzaldehyde headgroup harboring an unusual polyfunctional tail and an O-linked disaccharide comprising a 3-α-epimycarose and 2-O-methyl-α-rhamnose whose installation requires flavin monooxygenase-dependent hydroxylation of the polyketide product. Production of the fully decorated glycolipid was verified in cultures of two patient-derived Nocardia species. In both E. coli and Nocardia spp., the glycolipid was only detected in culture supernatants, consistent with data from genetic knockout experiments implicating roles for two dedicated proteins in installing the second sugar substituent only after the monoglycosyl intermediate is exported across the bacterial cell membrane. With the NOCAP product in hand, the stage is set for investigating the evolutionary benefit of this polyketide biosynthetic pathway for Nocardia strains capable of infecting human hosts.

Properties of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase.

Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all-trans-configured triene moiety in the polyketide product. Here, we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the "split-and-stuttering" module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase.

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

Dynamics and Microstructures of Nicotine/Water Binary Mixtures near the Lower Critical Solution Temperature.

The orientational dynamics and microscopic structures of nicotine/water binary mixtures near the system's lower critical solution temperature (LCST) were elucidated using optical heterodyne-detected optical Kerr effect (OHD-OKE) spectroscopy, nuclear magnetic resonance correlation spectroscopy (NMR COSY), first-principles calculations, and molecular dynamics simulations. Water concentrations were investigated from zero to close to pure water. At temperatures below the LCST, OHD-OKE experiments measured an anomalous slowing as the phase transition concentration was approached. At moderate concentrations and low temperatures, intermolecular cross-peaks between nicotine and water molecules were observed in the COSY spectra, demonstrating the formation of structures that persist for milliseconds. These results suggest that pair correlations contribute to the slowdown in the OHD-OKE data at moderate water concentrations. First-principles calculations revealed that intermolecular hydrogen bonding coordination between nitrogen atoms in pyridine moieties and water lowers the energy barriers for the reorientations of the two nicotine rings. Atomistic simulations demonstrate that with increasing water concentration, hydrogen bonding interactions between pyridine moieties and water molecules first increase and then decrease with a maximum at moderate water concentrations. These experimental and computational characterizations of the dynamics of nicotine molecules are attributed to the distinct configurations of water molecules around the pyridine ring moieties in nicotine molecules.

An Alkaloid from Scorpion Venom: Chemical Structure and Synthesis.

While most scorpion venom components identified in the past are peptidic or proteinic in nature, we report here a new alkaloid isolated from the venom of the Mexican scorpion Megacormus gertschi. Nuclear magnetic resonance and mass spectrometric investigations elucidate the structure of the alkaloid as ( Z)- N-(2-(1 H-imidazol-4-yl)ethyl)-3-(4-hydroxy-3-methoxyphenyl)-2-methoxyacrylamide (1). A chemical method of synthesizing this alkaloid is also described. Although abundant in venom, the above alkaloid was not found to have insecticidal activity. Structural analysis suggests that this venom alkaloid might be of potential interest for evaluating its medicinal effect.

Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis.

Although a few well-characterized polyketide synthases (PKSs) have been functionally reconstituted in vitro from purified protein components, the use of this strategy to decode "orphan" assembly line PKSs has not been described. To begin investigating a PKS found only in Nocardia strains associated with clinical cases of nocardiosis, we reconstituted in vitro its five terminal catalytic modules. In the presence of octanoyl-CoA, malonyl-CoA, NADPH, and S-adenosyl methionine, this pentamodular PKS system yielded unprecedented octaketide and heptaketide products whose structures were partially elucidated using mass spectrometry and NMR spectroscopy. The PKS has several notable features, including a "split, stuttering" module and a terminal reductive release mechanism. Our findings pave the way for further analysis of this unusual biosynthetic gene cluster whose natural product may enhance the infectivity of its producer strains in human hosts.

Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification.

N(6)-Methyladenosine (m(6)A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m(6)A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m(6)A-U pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred syn geometry on the Watson-Crick face to the higher-energy anti conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m(6)A in duplexes reveal that it is destabilizing by 0.5-1.7 kcal/mol. In contrast, we show that m(6)A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base.

Fast dynamics of HP35 for folded and urea-unfolded conditions.

The changes in fast dynamics of HP35 with a double CN vibrational dynamics label (HP35-P(2)) as a function of the extent of denaturation by urea were investigated with two-dimensional infrared (2D IR) vibrational echo spectroscopy. Cyanophenylalanine (PheCN) replaces the native phenylalanine at two residues in the hydrophobic core of HP35, providing vibrational probes. NMR data show that HP35-P(2) maintains the native folded structure similar to wild type and that both PheCN residues share essentially the same environment within the peptide. A series of time-dependent 2D IR vibrational echo spectra were obtained for the folded peptide and the increasingly unfolded peptide. Analysis of the time dependence of the 2D spectra yields the system's spectral diffusion, which is caused by the sampling of accessible structures of the peptide under thermal equilibrium conditions. The structural dynamics become faster as the degree of unfolding is increased.

Orientational and translational dynamics of polyether/water solutions.

Optical heterodyne-detected optical Kerr effect (OHD-OKE) experiments and pulsed field-gradient spin-echo NMR (PFGSE-NMR) experiments were performed to measure the rotational and translational diffusion constants of a polyether, tetraethylene glycol dimethyl ether (TEGDE), in binary mixtures with water over concentrations ranging from pure TEGDE to approaching infinite dilution. In addition, hydrodynamic calculations of the rotational and translational diffusion constants for several rigid TEGDE conformations in the neat liquid and in the infinitely dilute solution were performed to supplement the experimental data. The rotational relaxation data follow the Debye-Stokes-Einstein (DSE) equation within experimental error over the entire water concentration range. The agreement with the DSE equation indicates that there is no significant structural change of the polyether as the water content is changed. In contrast to the rotational dynamics, the translational diffusion data show a distinct deviation from Stokes-Einstein (SE) behavior. As the water content of the mixture is reduced, the translational diffusion rate decreases less rapidly than the increase in viscosity alone predicts until the water/TEGDE mole ratio of 7:1 is reached. Upon further reduction of water content, the translational diffusion tracks the viscosity. Comparison of the translational data with the rotational data and the hydrodynamic computations shows that the translational dynamics cannot be explained by a molecular shape change and that the low water fraction solutions are the ones that deviate from hydrodynamic behavior. A conjecture is presented as a possible explanation for the different behaviors of the rotational and translational dynamics.

Structure and replication of yDNA: a novel genetic set widened by benzo-homologation.

In a functioning genetic system, the information-encoding molecule must form a regular self-complementary complex (for example, the base-paired double helix of DNA) and it must be able to encode information and pass it on to new generations. Here we study a benzo-widened DNA-like molecule (yDNA) as a candidate for an alternative genetic set, and we explicitly test these two structural and functional requirements. The solution structure of a 10 bp yDNA duplex is measured by using 2D-NMR methods for a simple sequence composed of T-yA/yA-T pairs. The data confirm an antiparallel, right-handed, hydrogen-bonded helix resembling B-DNA but with a wider diameter and enlarged base-pair size. In addition to this, the abilities of two different polymerase enzymes (Klenow fragment of DNA pol I (Kf) and the repair enzyme Dpo4) to synthesize and extend the yDNA pairs T-yA, A-yT, and G-yC are measured by steady-state kinetics studies. Not surprisingly, insertion of complementary bases opposite yDNA bases is inefficient due to the larger base-pair size. We find that correct pairing occurs in several cases by both enzymes, but that common and relatively efficient mispairing involving T-yT and T-yC pairs interferes with fully correct formation and extension of pairs by these polymerases. Interestingly, the data show that extension of the large pairs is considerably more efficient with the flexible repair enzyme (Dpo4) than with the more rigid Kf enzyme. The results shed light on the properties of yDNA as a candidate for an alternative genetic information-encoding molecule and as a tool for application in basic science and biomedicine.

Toward a designed, functioning genetic system with expanded-size base pairs: solution structure of the eight-base xDNA double helix.

We describe the NMR-derived solution structure of the double-helical form of a designed eight-base genetic pairing system, termed xDNA. The benzo-homologous xDNA design contains base pairs that are wider than natural DNA pairs by ca. 2.4 A (the width of a benzene ring). The eight component bases of this xDNA helix are A, C, G, T, xA, xT, xC, and xG. The structure was solved in aqueous buffer using 1D and 2D NMR methods combined with restrained molecular dynamics. The data show that the decamer duplex is right-handed and antiparallel, and hydrogen-bonded in a way analogous to that of Watson-Crick DNA. The sugar-phosphate backbone adopts a regular conformation similar to that of B-form DNA, with small dihedral adjustments due to the larger circumference of the helix. The grooves are much wider and more shallow than those of B-form DNA, and the helix turn is slower, with ca. 12 base pairs per 360 degrees turn. There is an extensive intra- and interstrand base stacking surface area, providing an explanation for the greater stability of xDNA relative to natural DNA. There is also evidence for greater motion in this structure compared to a previous two-base-expanded helix; possible chemical and structural reasons for this are discussed. The results confirm paired self-assembly of the designed xDNA system. This suggests the possibility that other genetic system structures besides the natural one might be functional in encoding information and transferring it to new complementary strands.

Solution structure of xDNA: a paired genetic helix with increased diameter.

We describe the structure in aqueous solution of an extended-size DNA-like duplex with base pairs that are approximately 2.4 A longer than those of DNA. Deoxy-lin-benzoadenosine (dxA) was employed as a dA analogue to form hydrogen-bonded base pairs with dT. The 10mer self-complementary extended oligodeoxynucleotide 5'-d(xATxAxATxATTxAT) forms a much more thermodynamically stable duplex than the corresponding DNA sequence, 5'-d(ATAATATTAT). NMR studies show that this extended DNA (xDNA) retains many features of natural B-form DNA, but with a few structural alterations due to its increased helical diameter. The results give insight into the structural plasticity of the natural DNA backbone and lend insight into the evolutionary origins of the natural base pairs. Finally, this structural study confirms the hypothesis that extended nucleobase analogues can form stable DNA-like structures, suggesting that alternative genetic systems might be viable for storage and transfer of genetic information.

A four-base paired genetic helix with expanded size.

We describe a new molecular class of genetic-pairing system that has a native DNA backbone but has all four base pairs replaced by new, larger pairs. The base pairs include size-expanded analogs of thymine and of adenine, both extended by the width of a benzene ring (2.4 A). The expanded-diameter double helices are more thermodynamically stable than the Watson-Crick helix, likely because of enhanced base stacking. Structural data confirm a right-handed, double-stranded, and base-paired helical form. Because of the larger base size, all the pairs of this helical system are fluorescent, which suggests practical applications in detection of natural DNA and RNA. Our findings establish that there is no apparent structural or thermodynamic prohibition against genetic systems having sizes different from the natural one.

Comparison of X-ray crystal structure of the 30S subunit-antibiotic complex with NMR structure of decoding site oligonucleotide-paromomycin complex.

Aminoglycoside antibiotics that bind to 16S ribosomal RNA in the aminoacyl-tRNA site (A site) cause misreading of the genetic code and inhibit translocation. Structures of an A site RNA oligonucleotide free in solution and bound to the aminoglycosides paromomycin or gentamicin C1a have been determined by NMR. Recently, the X-ray crystal structure of the entire 30S subunit has been determined, free and bound to paromomycin. Distinct differences were observed in the crystal structure, particularly at A1493. Here, the NMR structure of the oligonucleotide-paromomycin complex was determined with higher precision and is compared with the X-ray crystal structure of the 30S subunit complex. The comparison shows the validity of both structures in identifying critical interactions that affect ribosome function.