Chemoenzymatic exchange of phosphopantetheine on protein and peptide

Abstract

Evaluation of new acyl carrier protein hydrolase (AcpH, EC 3.1.4.14) homologs from proteobacteria and cyanobacteria reveals significant variation in substrate selectivity and kinetic parameters for phosphopantetheine hydrolysis from carrier proteins. Evaluation with carrier proteins from both primary and secondary metabolic pathways reveals an overall preference for acyl carrier protein (ACP) substrates from type II fatty acid synthases, as well as variable activity for polyketide synthase ACPs and peptidyl carrier proteins (PCP) from non-ribosomal peptide synthases. We also demonstrate the kinetic parameters of these homologs for AcpP and the 11-mer peptide substrate YbbR. These findings enable the fully reversible labeling of all three classes of natural product synthase carrier proteins as well as full and minimal fusion protein constructs.

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering

“…10 The Pseudomonas aeruginosa AcpH is much more amenable in vitro , 65 facilitating its use in the laboratory to detach labeled cargo or recycling previously labeled carrier proteins. 66–68 …”

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis

“…Secondary structure analysis of the E. coli AcpH via Phrye2, ITassier, and RaptorX (Figure S10a–c), along with circular dichroism analysis performed on select homologs of AcpH, suggests that the AcpHs are primarily composed of α‐helices, with few to no β‐strand/sheet components . This is in stark contrast to PptH, whose α/β/β/α four‐layer sandwich fold consists of a roughly equal number of α‐helices and β‐strands, being composed ∼25.5% α‐helix, ∼5% 3 10 helix, and ∼16.2% β‐strand.…”

“…Initial studies confirmed the E. coli AcpH cleaves holo‐acyl carrier proteins (ACPs) in vitro and in vivo, confirming that the PpT hydrolase activity is physiologically relevant . Both the E. coli and P. aeruginosa AcpHs can hydrolyze a subset of native and nonnative holo‐ and acylated‐ACP substrates in vitro, hinting at a role in the termination of incorrectly assembled or otherwise malfunctional biosynthetic products and regulating holo‐ACP levels . Activity analysis in the presence and absence of metals confirmed the E. coli AcpH activity requires the presence of divalent metal cofactors, with a preference for Mn 2+ .…”

“…Gel‐filtration analysis of the E. coli and P. aeruginosa AcpHs indicated that the proteins are likely monomeric, and circular dichroism analysis of the P. aeruginosa , S. oneidenesis , and Cyanothece sp. PCC 7822 AcpH homologs revealed that the proteins are primarily composed of α‐helices . Issues in obtaining soluble protein upon expression ( E. coli AcpH) and the tendency to aggregate upon concentration ( P. aeruginosa AcpH) have precluded the in‐depth structural analysis of this enigmatic class of proteins …”

Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins

“…It might be hypothesised that PPTase-based transfer of Ac-CoA to the ACPs may only be an issue when the PKS is initially produced as apo-proteins. However the presence of ACP hydrolases in the cell, which catalyse the removal of the pantetheine moiety from ACPs [24][25][26] , may compete with the loading process of PPTases allowing further acetyl units to be loaded.…”