Application of a newly identified and characterized 18-o-acyltransferase in chemoenzymatic synthesis of selected natural and nonnatural bioactive derivatives of phoslactomycins

Abstract

Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays. Streptomyces sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of plmS(3) from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp plmS(3) open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in Escherichia coli and shown to be an 18-O acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (k(cat) of 28 +/- 3 min(-1) and K(m) of 88 +/- 16 microM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS(3) was shown to catalyze esterification of PLM G with CoA and N-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.

FIG. 1.

Proposed biosynthetic relationship between PLM products made by Streptomyces sp. strain HK 803. A cytochrome P450 monooxygenase (PlmS₂) catalyzes C-18 hydroxylation of PLM B to generate PLM G, which is subsequently 18-O acylated by PlmS₃.

FIG. 2.

Genetic strategy for generation of the NP8/pMSG1′ derivative of Streptomyces sp. strain HK 803. (A) Replacement of plmS₂-plmS₃ by aac(3)IV using a PCR targeting method. (B) Replacement of aac(3)IV in the plmS₂ expression plasmid (pMSG1) by aadA to generate pMSG1′.

FIG. 3.

HPLC analysis demonstrating PLM B production by the NP8 derivative of Streptomyces sp. strain HK 803 (top trace) and PLM G production by NP8/pMSG1′ (plmS₃ expression plasmid) (bottom trace).

FIG. 4.

HPLC chromatogram demonstrating PlmS₃-catalyzed esterification of PLM G (bottom trace) to yield PLM A (middle trace) and PLM C using isobutyryl-CoA and isovaleryl-CoA, respectively.

FIG. 5.

HPLC assay demonstrating PlmS₃-catalyzed generation of PLM G (top trace) by hydrolysis of a mixture of acylated PLMs (bottom trace). PLM B, which does not contain an acylated C-18-hydroxyl substituent, is present at the beginning and end of the incubation.

FIG. 6.

Proposed role for a common acyl-enzyme intermediate in the reactions catalyzed by PlmS₃.