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PubMed2002

Generation of multiple bioactive macrolides by hybrid modular polyketide synthases in Streptomyces venezuelae.

The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase, leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products.

Amino Acid Sequence↗
PubMed2002

The hidden steps of domain skipping: macrolactone ring size determination in the pikromycin modular polyketide synthase.

The pikromycin (Pik) polyketide synthase (PKS) from Streptomyces venezuelae comprises four multifunctional polypeptides (PikAI, PikAII, PikAIII, and PikAIV). This PKS can generate 12- and 14-membered ring macrolactones (10-deoxymethynolide and narbonolide, respectively) through the activity of its terminal modules (PikAIII and PikAIV). We performed a series of experiments involving the functional replacement of PikAIV in mutant strains with homodimeric and heterodimeric PikAIV modules to investigate the details of macrolactone ring size determination. The results suggest a new and surprising mechanism by which the penultimate hexaketide chain elongation intermediate is transferred from PikAIII ACP5 to PikAIV ACP6 before release by the terminal thioesterase domain. Elucidation of this chain transfer mechanism provides important new details about alternative macrolactone ring size formation in modular PKSs and contributes to the potential for rational design of structural diversity by combinatorial biosynthesis.

Anti-Bacterial Agents↗
PubMed2002

Modular NRPSs are monomeric.

NRPSs, PKSs, and hybrid NRPS/PKSs are modular proteins with similar assembly-line organizations. Although PKSs function as dimers, new data demonstrate that functional NRPSs are monomeric. This discovery has significant implications for engineering artificial assemblies for the production of novel biotherapeutics.

Bacteria↗
PubMed1996

A functional chimeric modular polyketide synthase generated via domain replacement.

BACKGROUND: Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. RESULTS: To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. CONCLUSIONS: These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

Multienzyme Complexes↗
PubMed1998

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis.

BACKGROUND: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

Amino Acid Sequence↗
PubMed1999

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Catalysis↗
PubMed2003

Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.

DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.

Amino Acid Sequence↗
PubMed2002

Modular design of artificial transcription factors.

Eukaryotic transcription factors are composed of interchangeable modules. This has led to the design of a wide variety of modular artificial transcription factors (ATFs) that can stimulate or inhibit the expression of targeted genes. The ability to regulate the expression of any targeted gene using a 'programmable' ATF offers a powerful tool for functional genomics and bears tremendous promise in developing the field of transcription-based therapeutics.

Biotechnology↗
PubMed2001

Modular complexes that regulate actin assembly in budding yeast.

The actin cytoskeleton of budding yeast contains an extensive set of actin-associated proteins with conserved mammalian counterparts. For more than 20 years, yeast has been used as a model organism to dissect the in vivo functions of these factors, revealing an intricate web of genetic interactions in the cell. Now, a surge of biochemical reports is defining the physical interactions and activities of these proteins and providing mechanistic insights into their cellular roles. The emerging view is that most actin-associated proteins do not act alone but, rather, associate to form modular protein complexes that regulate actin assembly and organization.

Actins↗
PubMed2000

Modular organization of excitatory and inhibitory reflex receptive fields elicited by electrical stimulation of the foot sole in man.

OBJECTIVES: The present study aimed to investigate how the inhibitory and excitatory reflex components of the human (polysynaptic) withdrawal reflex are organized depending on the stimulation site. The reflexes were elicited during a voluntary pre-contraction (between 10 and 20% of maximum voluntary contraction) of two antagonistic muscles. METHODS: Inhibitory and excitatory reflex receptive fields to tibialis anterior (TA) and soleus (SO) were mapped in 14 healthy subjects using randomized electrical stimulation at 16 sites of the foot sole. Low, non-painful (3x perception threshold), and high, painful (1.5x pain threshold), stimulus intensities were used. RESULTS: The inhibitory reflex receptive fields were organized in a highly functional manner supporting the action of the excitatory reflex. Together the two reflexes result in an optimal withdrawal from the stimulus. Low stimulation intensity was found sufficient to elicit the inhibitory reflex. High stimulation intensity caused a reversal of the inhibition to excitation in tibialis anterior. In soleus the inhibition was facilitated for stronger intensities. CONCLUSION: In conclusion, findings in animals of a modular organization of inhibitory reflexes are reproduced in humans.

Electric Stimulation↗
PubMed2004

Plasmodium vivax Duffy binding protein: a modular evolutionary proposal.

The population of malaria-causing parasites is characterized by great genetic diversity. Knowledge of the polymorphism generation mechanism is a central issue for developing effective vaccines against malaria and understanding the parasite population structure. Plasmodium vivax genetic diversity has been explained in terms of two major factors: natural selection and intragenic recombination. A modular organization was found within P. vivax Duffy binding protein in the present work. Four Colombian isolates have identical sequences to Salvador-1 strain amongst dpb regions III-VI analysed, suggesting a high identity between Central and South American isolates. Geographically clustered sectors, corresponding to cysteine-rich regions (II and VI), show a high sequence diversity that could reflect a possible immune response evasion mechanism; both positive and negative selection were detected in these regions. In contrast, other dbp gene regions display a non-geographical clustering pattern, lower sequence diversity and predominant negative selective pressure. Recombination was homogeneously detected all along the molecule. These findings suggest that diversification vs. homogenizing forces, drive dbp gene evolution and determine its mosaic region organization.

Amino Acid Sequence↗
PubMed2006

RNA structural motifs: building blocks of a modular biomolecule.

RNAs are modular biomolecules, composed largely of conserved structural subunits, or motifs. These structural motifs comprise the secondary structure of RNA and are knit together via tertiary interactions into a compact, functional, three-dimensional structure and are to be distinguished from motifs defined by sequence or function. A relatively small number of structural motifs are found repeatedly in RNA hairpin and internal loops, and are observed to be composed of a limited number of common 'structural elements'. In addition to secondary and tertiary structure motifs, there are functional motifs specific for certain biological roles and binding motifs that serve to complex metals or other ligands. Research is continuing into the identification and classification of RNA structural motifs and is being initiated to predict motifs from sequence, to trace their phylogenetic relationships and to use them as building blocks in RNA engineering.

Base Sequence↗
PubMed2000

Virtual lesions and the not-so-modular brain.

Because lesions usually affect contiguous areas of the brain, cognitive neuropsychology has generally posited modularity of brain function, assuming that specific regions of the brain have specific dedicated functions. However, a review of 36 published functional neuroimaging studies suggests that functions are distributed over multiple regions. Or, in other words, specific brain regions are usually multifunctional. By a technique of "virtual lesions," I measure the contribution that regions of the brain make to tasks imaged in 36 PET experiments, and propose a new and more detailed interlink between neuropsychology and functional neuroimaging.

Brain↗
PubMed2000

Modular phospholane ligands in asymmetric catalysis.

This Account outlines the preparation and application of a class of phosphine ligands based upon the trans-2,5-disubstituted phospholane moiety. The modular nature of these ligands has allowed facile variation of both phospholane substituent and backbone structure, thus providing access to a series of ligands. Bidentate bis(phospholane) ligands have been found to be very useful in asymmetric catalytic hydrogenation reactions. In particular, we highlight the versatility of highly efficient bis(phospholane)rhodium catalysts that allow enantioselective hydrogenation to produce a diverse range of compounds containing C-N, C-O, and C-C stereogenic centers.

Amides↗
PubMed2000

Phosphinooxazolines--a new class of versatile, modular P,N-ligands for asymmetric catalysis.

Chiral phosphinooxazolines (PHOX ligands), which coordinate to a metal center with a N- and a P-atom, allow effective enantiocontrol in a variety of metal-catalyzed reactions. They are readily synthesized, and because of their modular structure, the steric and electronic properties can be tailored for a specific application by variation of the oxazoline ring, the backbone, and the phosphine moiety.

Catalysis↗
PubMed1989

Modular design of synthetic protein mimics. Characterization of the helical conformation of a 13-residue peptide in crystals.

The incorporation of alpha-aminoisobutyryl (Aib) residues into peptide sequences facilitates helical folding. Aib-containing sequences have been chosen for the design of rigid helical segments in a modular approach to the construction of a synthetic protein mimic. The helical conformation of the synthetic peptide Boc-Aib-(Val-Ala-Leu-Aib)3-OMe in crystals is established by X-ray diffraction. The 13-residue apolar peptide adopts a helical form in the crystal with seven alpha-type hydrogen bonds in the middle and 3(10)-type hydrogen bonds at either end. The helices stack in columns, zigzag rather than linear, by means of direct NH...OC head to tail hydrogen bonds. Leucyl side chains are extended on one side of the helix and valyl side chains on the other side. Water molecules form hydrogen bonds with several backbone carbonyl oxygens that also participate in alpha-helix hydrogen bonds. There is no apparent distortion of the helix caused by hydration. The space group is P2(1)2(1)2(1), with a = 9.964 (3) A, b = 20.117 (3) A, c = 39.311 (6) A, Z = 4, and dx = 1.127 g/cm3 for C64H106N13O16.1.33H2O. The final agreement factor R was 0.089 for 3667 data observed greater than 3 sigma(F) with a resolution of 0.9 A.

Crystallization↗
PubMed2001

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA.

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.

Bacillus subtilis↗
PubMed2001

The C-terminal tail of the dual-specificity Cdc25B phosphatase mediates modular substrate recognition.

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases (Cdk/cyclins), thus triggering initiation and progression of successive phases of the cell cycle. In our efforts to elucidate the interaction between Cdc25B and the natural substrate, bis-phosphorylated Cdk2/CycA (Cdk2-pTpY/CycA), we have previously found that the 17 residues of the C-terminal tail mediate a factor of 10 in substrate recognition. In the studies reported here, we localize the majority of this interaction using site-directed mutagenesis to two arginine residues (Arg556 and Arg562) located within this C-terminal region. We also show that the catalytic domain of Cdc25C, which differs most significantly from Cdc25B in this tail region, has a 100-fold lower activity toward Cdk2-pTpY/CycA. We further demonstrate that the proper presentation of the C-terminal tail of Cdc25B can be achieved in a "gain-of-function" chimeric protein consisting of the C-terminal tail of Cdc25B fused onto the catalytic core of Cdc25C. The >10-fold increase in activity seen only in the chimeric protein containing the two critical arginine residues demonstrates that the modular C-terminal tail of Cdc25B is the basis for most of the catalytic advantage of Cdc25B versus Cdc25C toward the Cdk2-pTpY/CycA substrate.

Amino Acid Sequence↗