A Method for Prediction of the Locations of Linker Regions within Large Multifunctional Proteins, and Application to a Type I Polyketide Synthase

Abstract

Multifunctional proteins often appear to result from fusion of smaller proteins and in such cases typically can be separated into their ancestral components simply by cleaving the linker regions that separate the domains. Though possibly guided by sequence alignment, structural evidence, or light proteolysis, determination of the locations of linker regions remains empirical. We have developed an algorithm, named UMA, to predict the locations of linker regions in multifunctional proteins by quantification of the conservation of several properties within protein families, and the results agree well with structurally characterized proteins. This technique has been applied to a family of fungal type I iterative polyketide synthases (PKS), allowing prediction of the locations of all of the standard PKS domains, as well as two previously unidentified domains. Using these predictions, we report the cloning of the first fragment from the PKS norsolorinic acid synthase, responsible for biosynthesis of the first isolatable intermediate in aflatoxin production. The expression, light proteolysis and catalytic abilities of this acyl carrier protein-thioesterase didomain are discussed.

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

“…Recombinant PksA TE monodomain and PksA ACP-TE didomain constructs containing N-terminal His 6 -tags were produced and purified as previously described (7,9). The proteins were dialyzed against 50 mM Tris-HCl pH 7.5, and the polyhistidine tag was cleaved with bovine thrombin (10 unit per 1 mg sample).…”

“…In contrast to the chemically unreactive hydrocarbon chain generated in fatty acid biosynthesis, a poly-β-keto intermediate 5 is thought to be created by the nonreducing polyketide synthase (PKS) system. The second newly recognized domain, coined the product template (PT) domain (7), was shown to be critical in the biosynthetic process to control the correct cyclization of this highly reactive intermediate (9). In PksA, the KS, MAT, and ACP join with PT to extend the hexanoyl starter and carry out selective cyclization/aromatization chemistry (intermediate 6), which, either in the absence of TE, or with a nonfunctional TE gives rise to the pyrone 4, whereas in the presence of TE, a new C-C bond is formed to yield the Claisen product noranthrone 1 (Fig.…”

“…The PksA ACP-TE/CLC didomain (holoform) was previously shown to promote the hydrolysis of benzoyl N-acetylcysteamine thioester (benzoyl-SNAC; k cat 0.84 s −1 , K m 1.9 mM, and k cat ∕K m 0.44 s −1 mM −1 ) in vitro (7). While the N-acetyl cysteamine (NAC) thioester mimics part of the ACPphosphopantetheine (PPT) moiety that carries biosynthetic intermediates through the catalytic cycle, it is presumed that water acts as the nucleophile in the PksA TE/CLC domain to release the free acids in the absence of the native substrate.…”

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae

“…In contrast, PKS13 is a nonreducing PKS that does not contain any of the reductive tailoring domains. In addition to the minimal PKS, PKS13 contains an N-terminal region homologous to that of the starterunit-ACP transacylase (SAT) domain from the norsorlinic acid synthase (NSAS) (3), a putative product template (PT) domain (15), and a C-terminal thioesterase (TE) domain.…”

Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

“…A bioinformatics method, the Udwary-Merski algorithm (UMA), which predicts interdomainal linker regions across a family of related proteins by combining primary sequence similarity, predicted secondary structure, and local hydrophobicity has revealed in fungal, nonreducing PKSs two previously unrecognized and well delineated domains (19). The first is an extended N-terminal domain, and the second is an Ϸ350-aa central region of undetermined function.…”