Extender Unit Promiscuity and Orthogonal Protein Interactions of an Aminomalonyl-ACP Utilizing Trans-Acyltransferase from Zwittermicin Biosynthesis

Abstract

Trans-acting acyltransferases (trans-ATs) are standalone enzymes that select and deliver extender units to polyketide synthase assembly lines. Accordingly, there is interest in leveraging trans-ATs as tools to regioselectively diversify polyketide structures. Yet, little is known regarding the extender unit and acyl carrier protein (ACP) specificity of trans-ATs, particularly those that utilize unusual ACP-linked extender units. For example, the biosynthesis of the antibiotic zwittermicin involves the trans-AT ZmaF, which is responsible for installing a rare ACP-linked aminomalonyl extender unit. Here, we developed a method to access a panel of non-natural and non-native ACP-linked extender units and used it to probe the promiscuity of ZmaF, revealing one of the most promiscuous ATs characterized to date. Furthermore, we demonstrated that ZmaF is highly orthogonal with respect to its ACP specificity, and the ability of ZmaF to trans-complement noncognate PKS modules was also explored. Together, these results set the stage for further engineering ZmaF as a tool for polyketide diversification.

Figure 1.. ZMA biosynthesis by the PKS/NRPS hybrid pathway.

Each module is shown with the cognate building block tethered to the carrier protein. The pathway produces two metabolites (not shown), one of which is a prodrug that is cleaved to yield the active product, ZMA. The trans-AT ZmaF transfers an AmM extender unit to ZmaA-ACP1 through an ACP-linked extender unit, AmM-ZmaH. The subsequent PKS module also utilizes an ACP-linked extender unit (hydroxymalonyl-ZmaD) through the cis-AT, ZmaA-AT.

Figure 2.. Generation and analysis of ZmaH-linked extender units.

(A) An acyl-CoA panel was generated using MatB or suitable MatB mutant. The formation of these intermediates was confirmed by HPLC-UV analysis and used in the subsequent Sfp reaction to convert apo-ZmaH to acyl-ZmaH. Following the confirmation of acyl-ZmaH formation by MS, each acyl-ZmaH substrate was assayed with ZmaF in the presence of holo-ZACP1. Formation of acyl-ZACP1 was monitored via intact protein MS. (B) In order to assay ZmaF with its natural substrate, AmM-ZmaH, the enzymes involved in ZMA biosynthesis were employed to generate AmM-ZmaH in vitro from holo-ZmaH. (C) As an example, mass spectra showing conversion of apo-ZmaH to ethylmalonyl-ZmaH (EM-ZmaH) are shown. Purified apo-ZmaH (bottom) is incubated with Sfp and EM-ZmaH to generate EM-ZmaH. Holo-ZmaH is also generated as a result of remaining CoA from the MatB reaction used in the subsequent Sfp reaction. Gluconylated protein is present due to either spontaneous or enzymatic in vivo modification in E. coli.

Figure 3.. Activity of ZmaF with ZmaH-linked extender units.

(A) The ability of ZmaF to convert apo-ZACP1 to the corresponding acyl-ZACP1 with a panel of acyl-ZmaH was probed by intact protein MS. (B) Analysis of representative set of reactions to monitor ZmaF activity. EM-ZmaH was incubated with ZACP1 in the absence (top) and presence (bottom) of ZmaF to monitor ZmaF-dependent trans-acylation of ZACP1 to form EM-ZACP1. * gluconyl-holo-ZmaH; ** acetyl-holo-ZACP1 (C) Substrate specificity of ZmaF. Error bars represent standard deviation of duplicates performed.

Figure 4.

Azido-functionalized extender units as probes for ZmaF activity. (A) Thioester-linked azidoethylmalonates were tested with ZmaF and ZACP1. Following reaction with ZmaF, each product mixture was incubated with DBCO to fluorescently label any protein modified with the azide. (B) Each reaction was analyzed via SDS-PAGE gel followed by fluorescence-imaging. Signals on the fluorescence-imaged gel (top) indicate that the protein has been acylated with AzEM. The gel was then subjected to Coomassie staining (bottom) to visualize all proteins in the reaction.

Figure 5.. Probing the orthogonality of the ZMA ATs.

The ability of each substrate (acyl-ZmaH or acyl-ZmaD) and enzyme (ZmaF or ZmaA-AT) pair to trans-acylate each ACP (ZACP1 or ZACP2) was determined using intact protein MS. To determine the ability of each enzyme (ZmaF or ZmaA-AT) to undergo self-acylation with AzEM-ZmaH or AzEM-ZmaD (R=AzEM), transfer of the azide to the AT active site was determined by in-gel fluorescence.

Figure 6.. Docking studies with homology models using ClusPro.

(A) Surface view of ZmaF to visualize the active site chamber. The active site serine is yellow and circled. (B) The top three docking locations of ZmaH. (C) The top three selected docking locations of ZACP1. The Ppant attachment sites of each ACP structure are shown as yellow spheres. In all cases, for clarity, the poorly resolved C-terminus of ZmaF was removed from the structure.

Figure 7.

Trans-complementation assays of DEBS modules. (A) General reaction scheme showing the use of DKS and a CoA- or ACP-linked extender unit, to probe the ability of trans-ATs to complement a DEBS module. (B) Summary of the key results obtained from the trans-complementation assays.