Transthioesterification

Figure 17.13 Expressed proteins containing a thioester intein tag can be specifically modified using a cysteine-alkyne derivative by transthioesterification followed by an internal S - N shift.

Figure 17.26 The native process leading to intein excision and ligation of extein fragments involves a sequence of reactions involving transthioesterification, cleavage of the intein fragment, and aS->N shift, which ligates the two extein peptides together via an amide bond.

The ability of DNA to serve as a template for the reversible formation of a PNA oligomer was reported by the Ghadiri group in 2009 [44]. The overall framework here relied on the use of a transthioesterification reaction as the exchange mechanism, operating between derivatives of DNA bases (for example, the adenine... 

Fig. 18 Transthioesterification reaction used by Ghadiri and coworkers in the dynamic production of DNA-templated PNAs...

In 2004, Ramstrom and coworkers reported the first prototype DCLs based on a reversible transthioesterification reaction [47]. DCLs of potentially ten different thioesters were generated from a series of five thioesters - all prepared from 3-sulfanylpropionic acid - and one thiol (thiocholine). Transthioesterification (exchange) reactions were observed by simply mixing the different DCL components in aqueous solution. Interestingly, when added to the equilibrated mixture of thioesters, the enzyme acetylcholinesterase was able to recognize and subsequently hydrolyze its best substrate selectively. This selective hydrolysis... 

Figure 1 Native chemical ligation (NCL) between two unprotected peptide segments. The initial transthioesterification reaction leads to an intermediate that undergoes an S to N-acyl shift via a five-membered cyclic transition state and generates a native amide bond at the ligation site.

Figure 4 Mechanism of trans-protein splicing, (a) Initial association of the intein halves to form a functional intein. (b) Activation of the N-terminal splice-junction via an N-S acyl shift, (c) Formation of a branched intermediate upon transthioesterification. (d) Branch resolution and intein release by succinimide formation. Spontaneous S-N acyl rearrangement yields the processed product with a native peptide backbone.

KR), dehydratase (DH), and enol reductase (ER) domains while docked at the ACP. Once fully processed, the extended chain is passed to the KS of the subsequent module by a transthioesterification reaction. The process is repeated, leading to the final module where the product is generally excised via hydrolysis or thioesterase (TE) mediated macrocyclization. 

The less clearly understood aromatic PKSs utilize a single KS(CLF)/ACP pair capable of multiple elongation reactions to construct the complete polyketide backbone. The number of elongation events is controlled by the CLF associated with the KS domain. Transthioesterification and decarboxylative condensation reactions proceed in an analogous fashion to modular systems. The ultimate topology of advanced aromatic polyketides is controlled by precise combination of tailoring enzymes responsible for redox chemistry and cyclization pattern. 

The first enzyme in the system catalyzes the reaction of TPP with pyruvate to form the same resonance-stabilized carbanion formed by pyruvate decarboxylase and by the enzyme in Problems 8 and 9. The second enzyme of the system (E2) requires lipoate, a coenzyme that becomes attached to its enzyme by forming an amide with the amino group of a lysine side chain. The disulfide linkage of lipoate is cleaved when it undergoes nucleophilic attack by the carbanion. In the next step, the TPP carbanion is eliminated from the tetrahedral intermediate. Coenzyme A (CoASH) reacts with the thioester in a transthioesterification reaction (one thioester is converted into another), substituting coenzyme A for dihydrolipoate. At this point, the final reaction product (acetyl-CoA) has been formed. However, before another catalytic cycle can occur, dihydrolipoate must be oxidized back to lipoate. This is done by the third enzyme (E3), an FAD-requiring enzyme (Section 25.3). Oxidation... 

Scheme 32 Transthioesterification of 2-thiol Trp and subsequent S — IV acyl shift (path a) or interception with exogenous thiophenol (path b)...

Keywords Ligation N-S acyl shift Peptide thioester modification Protein synthesis Transthioesterification... 

---