Modifications of Protein-Bound Substrates by Trans-Acting Enzymes in Natural Products Biosynthesis

Abstract

Enzymatic modifications of small molecules are a common phenomenon in natural product biosynthesis, leading to the production of diverse bioactive compounds. In polyketide biosynthesis, modifications commonly take place after the completion of the polyketide backbone assembly by the polyketide synthases and the mature products are released from the acyl-carrier protein (ACP). However, exceptions to this rule appear to be widespread, as on-line hydroxylation, methyl transfer, and cyclization during polyketide assembly process are common, particularly in trans-AT PKS systems. Many of these modifications are catalyzed by specific domains within the modular PKS systems. However, several of the on-line modifications are catalyzed by stand-alone proteins. Those include the on-line Baeyer-Villiger oxidation, α-hydroxylation, halogenation, epoxidation, and methyl esterification during polyketide assembly, dehydrogenation of ACP-bound short fatty acids by acyl-CoA dehydrogenase-like enzymes, and glycosylation of ACP-bound intermediates by discrete glycosyltransferase enzymes. This review article highlights some of these trans-acting proteins that catalyze enzymatic modifications of ACP-bound small molecules in natural product biosynthesis.

Keywords: acyl carrier protein; on-line modification; polyketide synthase; tailoring reaction; trans-acting enzyme.

Figure 1.

Examples of on-line modification of polyketides by discrete enzymes. (a) cycloether formation by SalBIII in salinomycin biosynthesis; (b) cyclopropanation by Jaw5 in jawsamycin biosynthesis; and (c) intramolecular [4+2] cycloaddition by CghA in Sch 210972 biosynthesis.

Figure 2.

On-line modifications in colibactin and barbamide biosyntheses. (a) The use of S-adenosylmethionine as a building block and on-line cyclopropanation in the colibactin assembly line and (b) on-line α-ketothioester decarboxylation by a ketosynthase domain in barbamide biosynthesis.

Figure 3.

Proposed biosynthetic pathway to oocydin B. The trans-AT PKS system is highly versatile with several on-line modifications, e.g., Baeyer-Villiger oxidation (purple), α-hydroxylation (green), halogenation (aqua), and acylation (orange). OocC-G (highlighted in grey) is a β-branching cassette, which introduces methyl groups to the nascent polyketide chains. OocP is a stand-alone halogenase, whereas OocQ is an unknown protein that is proposed to stabilize an OocPQ complex.

Figure 4.

Proposed biosynthetic pathway to oximidines. (a) Two flavin-dependent monooxygenase domains, LbmA-Ox and LbmC-Ox are proposed to catalyze the formation of the oxime (aqua), whereas the LbmC-Ox domain catalyzes the Baeyer-Villiger-type oxygen-insertion into the nascent polyketide chain (pink). (b) A flavin-dependent monooxygenase domain, OxiB-Ox, is proposed to catalyze the formation of the oxime (aqua), where the P₄₅₀ OxiK is proposed to install the epoxide post-assembly.

Figure 5.

On-line methyl esterification in aurantinin biosynthesis by the SAM-dependent protein Art28. The methyl ester is retained until the last step of the pathway, where the esterase Art9 hydrolyzes the methyl ester and converts the inactive aurantinin 9B to the active aurantinin B.

Figure 6.

Epoxidation of an ACP-bound polyketide intermediate in shuangdaolide biosynthesis followed by the proposed chemical cascade that results in the transformation of shuangdaolide Q2 to ashuangdaolide Q1.

Figure 7.

Chemical structures of several natural products with a terminal alkene.

Figure 8.

Variants of acyl-CoA dehydrogenases are involved in the formation of the terminal akenes in FK506 and haliangicin biosyntheses. (a) The γ,δ-variant of acyl-CoA dehydrogenase TcsD is involved in the formation of allylmalonyl-CoA, an extender unit in FK506 biosynthesis. An earlier study suggested that the crotonyl-CoA reductase TcsC precedes TcsD (gray bold arrows). A more recent study showed that TscD precedes TcsC (black bold arrows); (b) The γ,δ-acyl-CoA dehydrogenase variant HliR is involved in haliangicin biosynthesis.

Figure 9.

ACP-dependent dehydrogenation involved in the biosynthesis of NFAT-133 and its analogues. (a) Chemical structures of NFAT-133 and its analogues; (b) A variant of acyl-CoA dehydrogenase, NftN, may catalyze the conversion of 2-pentenoyl-ACP to 2,4-pentadienoyl-ACP during NFAT-133 polyketide assembly.

Figure 10.

Glycosylation of ACP-bound polyketide intermediate and subsequent modifications in pactamycin biosynthesis.

Figure 11.

Glycosylation of ACP-bound AHBA and subsequent modification in mitomycin biosynthesis. (a) attachment of AHBA to the acyl carrier protein MmcB and glycosylation by the glycosyltransferase MitB; (b) deacetylation of GlcNAc-AHBA-MmcB to GlcN-AHBA-MmcB and transformation of the sugar moiety to a linear aminodiol that terminates with an epoxyethane catalyzed by the NADPH-dependent protein MitF and the radical SAM protein MitD.