Combinatorial Manipulation Research Articles

Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003

Nonribosomal peptide synthetases (NRPSs) catalyze the assembly of a structurally diverse group of peptides by the multiple-carrier thiotemplate mechanism. All NRPSs known to date are exclusively type I modular enzymes that consist of domains, such as adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains, for individual enzyme activities. Although several A and PCP domains have been demonstrated to function independently, aminoacylation in trans has been successful only between PCPs and their cognate A domains. We have identified within the bleomycin-biosynthesis gene cluster from Streptomyces verticillus ATCC15003 the blmI gene that encodes a discrete PCP protein. We overexpressed the blmI gene in Escherichia coli, purified the BlmI protein, and demonstrated that apo-BlmI can be efficiently modified into holo-BlmI either in vivo or in vitro by PCP-specific 4'-phosphopantetheine transferases (PPTases). Unlike the PCP domains in type I NRPSs, BlmI lacks its cognate A domain and can be aminoacylated by Val-A, an A domain from a completely unrelated type I NRPS. BlmI represents the first characterized type II PCP. The BlmI type II PCP, like the PCP domains of type I NRPSs, can be 4'-phospho-pantetheinylated by PCP-specific PPTases but is biochemically distinct in that it can be aminoacylated by an A domain from a completely unrelated type I NRPS. Our results provide for the first time the genetic and biochemical evidence to support the existence of a type II NRPS, which might be useful in the combinatorial manipulation of NRPS proteins to generate novel peptides.

Tolerance and specificity of polyketide synthases.

Polyketide synthases catalyze the assembly of complex natural products from simple precursors such as propionyl-CoA and methylmalonyl-CoA in a biosynthetic process that closely parallels fatty acid biosynthesis. Like fatty acids, polyketides are assembled by successive decarboxylative condensations of simple precursors. But whereas the intermediates in fatty acid biosynthesis are fully reduced to generate unfunctionalized alkyl chains, the intermediates in polyketide biosynthesis may be only partially processed, giving rise to complex patterns of functional groups. Additional complexity arises from the use of different starter and chain extension substrates, the generation of chiral centers, and further functional group modifications, such as cyclizations. The structural and functional modularity of these multienzyme systems has raised the possibility that polyketide biosynthetic pathways might be rationally reprogrammed by combinatorial manipulation. An essential prerequisite for harnessing this biosynthetic potential is a better understanding of the molecular recognition features of polyketide synthases. Within this decade, a variety of genetic, biochemical, and chemical investigations have yielded insights into the tolerance and specificity of several architecturally different polyketide synthases. The results of these studies, together with their implications for biosynthetic engineering, are summarized in this review.

Manipulative Tendencies of Captive Cebus albifrons

We conducted two experiments to examine the manipulative tendencies of captive Cebus albifrons. In Experiment 1 we examined hand preference for reaching by providing subjects with food either on the cage floor (to facilitate quadrupedal reaching) or at the height of an upright subject's shoulder (to facilitate bipedal reaching). In Experiment 2 we examined combinatorial manipulation by providing subjects with nesting containers and other portable manipulanda. Results indicate that C. albifrons exhibits greater use of the right hand for bipedal versus quadrupedal reaching (exhibiting a group-level lack of bias for bipedal reaching and a left-hand bias for quadrupedal reaching), combines objects using a simple pairing strategy, and uses and produces simple tools. Aspects of these findings parallel those for Cebus apella.

Harnessing the biosynthetic code: combinations, permutations, and mutations.

Polyketides and non-ribosomal peptides are two large families of complex natural products that are built from simple carboxylic acid or amino acid monomers, respectively, and that have important medicinal or agrochemical properties. Despite the substantial differences between these two classes of natural products, each is synthesized biologically under the control of exceptionally large, multifunctional proteins termed polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) that contain repeated, coordinated groups of active sites called modules, in which each module is responsible for catalysis of one complete cycle of polyketide or polypeptide chain elongation and associated functional group modifications. It has recently become possible to use molecular genetic methodology to alter the number, content, and order of such modules and, in so doing, to alter rationally the structure of the resultant products. This review considers the promise and challenges inherent in the combinatorial manipulation of PKS and NRPS structure in order to generate entirely "unnatural" products.

Engineered intermodular and intramodular polyketide synthase fusions

Background: Modular polyketide synthases (PKSs) are very large multifunctional enzyme complexes that synthesize a number of medicinally important natural products. The modular arrangement of active sites has made these enzyme systems amenable to combinatorial manipulation for the biosynthesis of novel polyketides. Here, we investigate the involvement of subunit interactions in hybrid and artificially linked PKSs with several series of intermodular and intramodular fusions using the erythromycin (6-deoxyerythronolide B synthase; DEBS) and rapamycin (RAPS) PKSs. Results: Several two-module and three-module derivatives of DEBS were constructed by fusing module 6 to either module 2 or module 3 at varying junctions. Polyketide production by these intramodular fusions indicated that the core set of active sites remained functional in these hybrid modules, although the ketoreductase domain of module 6 was unable to recognize unnatural triketide and tetraketide substrates. Artificial trimodular PKS subunits were also engineered by covalently linking modules 2 and 3 of DEBS, thereby demonstrating the feasibility of constructing single-chain PKSs. Finally, a series of fusions containing DEBS and RAPS domains in module 2 of an engineered trimodular PKS revealed the structural and functional tolerance for hybrid modules created from distinct PKS gene clusters. Conclusions: The general success of the intermodular and intramodular fusions described here demonstrates significant structural tolerance among PKS modules and subunits and suggests that substrate specificity, rather than protein-protein interactions, is the primary determinant of molecular recognition features of PKSs. Furthermore, the ability to artificially link modules may considerably simplify the heterologous expression of modular PKSs in higher eukaryotic systems.

MULTI-PLAQUETTE SOLUTIONS FOR DISCRETIZED ASHTEKAR GRAVITY

A discretized version of canonical quantum gravity proposed by Loll is investigated. After slightly modifying Loll’s discretized Hamiltonian constraint, we encode its action on the spin network states in terms of combinatorial topological manipulations of the lattice loops. Using this topological formulation we find new solutions to the discretized Wheeler-DeWitt equation. These solutions have their support on the connected set of plaquettes. We also show that these solutions are not normalizable with respect to the induced heat-kernel measure on SL(2, C) gauge theories.

Hierarchical complexity of combinatorial manipulation in capuchin monkeys (Cebus apella).

The purpose of this study was to examine the hierarchical complexity of combinatorial manipulation in capuchin monkeys (Cebus apella). Two experiments were conducted. In Experiment 1 capuchins were presented with an apparatus designed to accommodate the use of probing tools. In Experiment 2 the same capuchins were presented with sets of nesting containers. Five of the ten subjects used probing tools and seven subjects placed objects in the containers. The capuchins' behavior reflected three hierarchically organized combinatorial patterns displayed by chimpanzees and human infants. Although the capuchins sometimes displayed the two more complex patterns ("pot" and "subassembly"), their combinatorial behavior was dominated by the simplest pattern ("pairing"). In this regard capuchins may not attain the same grammar of manipulative action that has been reported for chimpanzees and young human children. © 1994 Wiley-Liss, Inc.

Object manipulation and the use of tools by infant baboons (Papio cynocephalus anubis).

Infant baboons (Papio cynocephalus anubis) in a captive peer group used objects as containers, drinking utensils, and sponges in the context of play. The baboons later used paper, browse, and other materials as tools to extract sweet liquids from apparatus designed to accommodate sponging and probing behavior. The results of this study demonstrate flexible combinatorial manipulation and spontaneous use of tools by infant baboons. These data are consistent with hypotheses that (a) an evolutionary history of omnivorous extractive foraging is associated with the use of tools and (b) free play in an object-enriched captive environment may facilitate combinatorial manipulation in nonhuman primates.

Computer-encouraged serendipity in pure mathematics

Computer applications to research in pure mathematics during the past several years have concentrated on using the data processing and logical capabilities of computers to carry out exhaustive and exhausting combinatorial or logical manipulations. Computers can also be used to suggest entirely new theorems and results, but this sort of application has not yet been particularly widely investigated. The addition of an interactive graphics terminal to even a small-to medium-sized computer enables one to construct an "n-dimensional blackboard." On this device a mathematician can display and manipulate two-dimensional projections of n-dimensional geometrical constructs with much greater speed and accuracy than he can bring to his usual blackboard sketches. This sort of facility can be particularly helpful to the type of mathematician who thinks geometrically, and who relies a great deal on geometrical visualizations to suggest new theorems and proofs. Using control dials, joysticks, and other analog input devices, a mathematician can get immediate portrayal of the geometric effect of continuously varying parameters. He also has finger-tip control over the current values of these parameters, encouraging the development of a visceral feeling for the effect of these parameter variations. Early experience with such an "n-dimensional blackboard," implemented in a prototype version on the microprogrammed minicomputer BUGS system at Brown University, is extremely promising. This BUGS blackboard, used in the area of singularities of algebraic curves in complex two-space, has suggested several new theorems and areas for further investigation. A description is given of the facilities of the BUGS blackboard. Also, the observed interaction between the physical facilities available on the BUGS system is mentioned, as well as the sorts of mathematical results that the use of these facilities in the BUGS blackboard seems to encourage.