Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module

Abstract

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

Nature's Combinatorial Biosynthesis Produces Vatiamides A–F

“…[1] In cyanobacteria, the study of saxitoxin, cylindrospermopsin, curacin A, and apratoxin A pathways,a mong others,h as provided much information on protein-protein interactions,n ovel gene function, and loading-module diversity in NP biosynthesis. [2][3][4][5][6][7][8] In typical PKS/ NRPS systems,a ctivated acyl-CoAs and amino acids are sequentially coupled by individual PKS or NRPS modules. This occurs collinearly such that the order in which the genes are transcribed is coincident with the functioning of proteins that selectively interact to elongate NPs with high fidelity.The interface between modules occurs via interacting type Io rII docking domains (dd) between PKSs and communication (COM) domains between some NRPS modules.…”

The Chemistry, Biochemistry and Pharmacology of Marine Natural Products from Leptolyngbya, a Chemically Endowed Genus of Cyanobacteria

“…collected from Palmyra Atoll in the Central Pacific Ocean ( Figure 11 ). Palmyrolide A ( 40 ) comprises a rare N -methyl enamide functionality, as well as an intriguing t -butyl branch that likely results biosynthetically from the incorporation of malonate and three methyl groups from S -adenosyl- l -methionine (SAM), as shown for apratoxin [ 45 , 65 ]. The absolute configuration of this molecule was assigned by total synthesis of both the natural (−)-palmyrolide A ( 40 ) and its enantiomer, (+)-ent-palmyrolide A.…”

Nature's Combinatorial Biosynthesis Produces Vatiamides A–F

“…Numerous microbial species contain polyketide synthase/non ribosomal peptide synthetase (PKS/NRPS) biosynthetic gene clusters, which produce diverse modified lipopeptide natural products (NP) . In cyanobacteria, the study of saxitoxin, cylindrospermopsin, curacin A, and apratoxin A pathways, among others, has provided much information on protein–protein interactions, novel gene function, and loading‐module diversity in NP biosynthesis . In typical PKS/NRPS systems, activated acyl‐CoAs and amino acids are sequentially coupled by individual PKS or NRPS modules.…”