Christopher T. Calderone

Directing Otherwise Incompatible Reactions in a Single Solution by Using DNA‐Templated Organic Synthesis

General methods for translating amplifiable information carriers such as DNA into synthetic molecules may enable the evolution of non-natural molecules through iterated cycles of translation, selection, and amplification that are currently available only to proteins and nucleic acids. During the process of developing such a method, we recently discovered that DNA templates can sequence-specifically direct a broad range of chemical reactions without any apparent structural requirements.[1,2] The generality of DNA-templated synthesis together with appropriate linker and purification strategies enabled the first multistep small-molecule syntheses programmed by DNA templates,[3] which raised the possibility of using this approach to generate synthetic small-molecule libraries of useful complexity. DNA-templated synthesis[4±24] can generate products individually linked to oligonucleotides that both encode and direct their syntheses.[1±3] This feature may enable reaction modes useful for library construction that are not available through current synthetic approaches. Present synthesis methodology, for example, cannot differentiate functional groups of similar reactivity on different molecules within the same solution even though such differentiation would enable diversification to take place without the effort or constraints associated with spatial separation. Here we report that DNA oligonucleotides can simultaneously direct several different types of synthetic reactions within the same solution, even though the reactants involved would be cross-reactive and therefore incompatible under traditional synthesis conditions. Our findings represent a new mode of reaction made possible by DNA-templated synthesis and may enable the one-pot diversification of synthetic library precursors into products of multiple reaction types. The ability of DNA templates to mediate diversification by using different types of reaction without spatial separation was first tested by preparing three oligonucleotide templates of different DNA sequences (1a±3a) functionalized at their 5’-ends with maleimide groups and three oligonucleotide reagents (4a±6a) functionalized at their 3’-ends with an amine, thiol, or nitroalkane group, respectively. The DNA sequences of the three reagents each contained a different 10base annealing region that was complementary to ten bases COMMUNICATIONS

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit α-hydroxyisocaproate (α-HIC). We show here that it arises from initial activation of α-ketoisocaproate (α-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The α-KIC unit is elongated to α-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the α-keto group of α-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the α-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function α- and β-KR acting on disparate positions of a growing chain intermediate.

FenF: Servicing the Mycosubtilin Synthetase Assembly Line in trans

Mycosubtilin biosynthesis is accomplished by a hybrid type I polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) assembly line. During polyketide biosynthesis, the assembly line utilizes an unusual free-standing acyltransferase (AT) enzyme, FenF, to supply the malonate extender unit for chain elongation. The in trans interaction between an AT domain and a type I PKS is unusual, as AT domains typically operate in cis with type I PKS. To better understand the interactions of in trans AT domains with type I PKS, we have completed kinetic and selectivity studies exploring the interactions between FenF and its ACP substrate. Mycosubtilin, an N-acyl cyclic Bacillus subtilis-derived octapeptide with anti-fungal activity, is assembled by a four-protein thiotemplate assembly line (Scheme 1). 2] The first protein in this assembly line is MycA, a 495 kDa polypeptide comprising eleven domains parceled into a two-domain fatty acid activating module, a PKS module, and a NRPS module. The fatty acid activating module contains an acyl-AMP ligase (AL) and an acyl carrier protein (ACP1) ; the PKS module includes a ketosynthase (KS) domain and ACP2, but lacks the third PKS “core” domain, an AT; the NRPS module has two peptidyl carrier proteins (PCP1 and PCP2) even though only a single amino acid (Asn) is thought to be activated by that module. Close inspection of the PKS module of MycA by bioinformatics analysis predicts the presence of a 100-residue stretch, termed a docking (D) domain (see below), between the KS and ACP2 domains. The three modules of MycA are proposed to act in tandem, combining palmitic acid, malonic acid (from malonyl CoA), and Asn to generate b-aminostearoyl-N-Asn1-S-PCP2 prior to transfer to MycB. The b-amino group of b-aminostearoyl-N-Asn1-SPCP2, generated by an aminotransferase (AMT) domain embedded within MycA, serves as the cyclizing nucleophile in heptapeptide macrolactam formation, as mycosubtilin biosynthesis is completed by the NRPS proteins MycB and MycC. The fourth protein in mycosubtilin biosynthesis is FenF, predicted to be a malonyl-selective AT domain acting in trans with MycA to deliver the C2 unit for palmitoyl chain elongation. [2]

Changes in motion vs. bonding in positively vs. negatively cooperative interactions

From a consideration of the interactions between non-covalent bonds, it is concluded that positively cooperative binding will occur with a benefit in enthalpy and a cost in entropy, and that negatively cooperative binding will occur with a cost in enthalpy and a benefit in entropy; experimental data support these conclusions.