Enzymatic basis of "hybridity" in thiomarinol biosynthesis

Abstract

Thiomarinol is a naturally occurring double-headed antibiotic that is highly potent against methicillin-resistant Staphylococcus aureus. Its structure comprises two antimicrobial subcomponents, pseudomonic acid analogue and holothin, linked by an amide bond. TmlU was thought to be the sole enzyme responsible for this amide-bond formation. In contrast to this idea, we show that TmlU acts as a CoA ligase that activates pseudomonic acid as a thioester that is processed by the acetyltransferase HolE to catalyze the amidation. TmlU prefers complex acyl acids as substrates, whereas HolE is relatively promiscuous, accepting a range of acyl-CoA and amine substrates. Our results provide detailed biochemical information on thiomarinol biosynthesis, and evolutionary insight regarding how the pseudomonic acid and holothin pathways converge to generate this potent hybrid antibiotic. This work also demonstrates the potential of TmlU/HolE enzymes as engineering tools to generate new "hybrid" molecules.

Keywords: antibiotics; biosynthesis; dithiolopyrrolone; enzymes; evolution.

Figure 1

A) Structures of thiomarinols, pseudomonic acids, and dithiopyrrolones. B) Gene cluster for thiomarinol. Open arrows indicate ORFs with homology to the mupirocin pathway; blue ORFs are homologous to DTP biosynthetic genes; black ORFs are unique to the thiomarinol pathway; red ORFs, TmlU and HolE, the targets of this study, have counterparts in the mupirocin and holomycin pathway, respectively. They are represented in red arrows instead of open and blue arrows for clarity.

Figure 2

Enzymatic production of PAC-holothin, a thiomarinol analogue, in vitro by TmlU and HolE, in the presence of 1 mM ATP, 2 mM MgCl₂, and 1 mM CoASH, at pH 7.5. A) Synthetic PAC-holothin standard, B) In vitro reconstitution of TmlU and HolE activity generating PAC-holothin, C) Control lacking CoA, D) Control lacking TmlU, E) Control lacking HolE, F) Mass spectrum of the PAC-CoA product generated by TmlU (calculated [M+H]⁺, 1234.4155). G) Mass spectrum of PAC-holothin product generated enzymatically by TmlU and HolE (calculated [M+H]⁺, 639.2768).

Figure 3

TmlU Kinetic Curves of different substrates. A) PAC, K_m = 6 ± 1 μM and k_cat = 3.2 ± 0.1 s⁻¹ B) PAA, K_m = 5.2 ± 0.5 μM, k_cat = 3.0 ± 0.1 s⁻¹ C) octanoic acid, K_m = 0.5 ± 0.1 mM and k_cat = (5.0 ± 0.3) × 10^−;3 s⁻¹.

Figure 4

Substrate promiscuity of TmlU and HolE. A) Carboxylic acids tested for incorporation into thiomarinol by 5 μM TmlU in the presence of saturating concentrations of HolE and holothin. Activity was measured by the formation of the acyl-holothin products at UV_{360 nm} and normalized to activity with PAC as substrate. [a] 1 μM TmlU was used in this assay. [b] 2,4-DDA, (2E,4E)-2,4-dodecadienoic acid. [c] 2,4-DA, (2E,4E)-2,4-decadienoic acid. B) Acyl-CoAs as substrates for HolE to generate acyl-holothin products. Activity was measured as described in A. C) Amines as substrates for HolE. Co-substrate PAC-CoA was generated by TmlU. Activity was measured by integration of ion intensities in mass spectra.

Figure 5

Mechanism of thiomarinol production by TmlU and HolE compared to assembly-line tethered mechanisms to create PKS/NRPS hybrids. A) Condensation domain of a NRPS directly adds a PKS product to a growing peptide chain. Chemistry occurs on the assembly line. B) A standalone transglutaminase-like domain (TGH) catalyzes the transfer between tethered PKS and NRPS products in the biosynthesis of andrimid.^[21] C) Tailoring enzymes TmlU and HolE create a NRPS/PKS hybrid with released products of discrete PKS and NRPS pathways. KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; C, condensation domain; A, adenylation domain; PCP, peptidyl carrier protein.

Scheme 1

A) Reported mechanism of amide formation catalysed by SimL.^[12] B) Our proposed mechanism of thiomarinol formation catalysed by TmlU/HolE.