Cloning of the Pactamycin Biosynthetic Gene Cluster and Characterization of a Crucial Glycosyltransferase Prior to a Unique Cyclopentane Ring Formation

Abstract

The biosynthetic gene (pct) cluster for an antitumor antibiotic pactamycin was identified by use of a gene for putative radical S-adenosylmethionine methyltransferase as a probe. The pct gene cluster is localized to a 34 kb contiguous DNA from Streptomyces pactum NBRC 13433 and contains 24 open reading frames. Based on the bioinformatic analysis, a plausible biosynthetic pathway for pactamycin comprising of a unique cyclopentane ring, 3-aminoacetophenone, and 6-methylsalicylate was proposed. The pctL gene encoding a glycosyltransferase was speculated to be involved in an N-glycoside formation between 3-aminoacetophenone and UDP-N-acetyl-a -D-glucosamine prior to a unique cyclopentane ring formation. The pctL gene was then heterologously expressed in Escherichia coli and the enzymatic activity of the recombinant PctL protein was investigated. Consequently, the PctL protein was found to catalyze the expected reaction forming b-N-glycoside. The enzymatic activity of the PctL protein clearly confirmed that the present identified gene cluster is for the biosynthesis of pactamycin. Also, a glycosylation prior to cyclopentane ring formation was proposed to be a general strategy in the biosynthesis of the structurally related cyclopentane containing compounds.

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

“…[11] However, there was no direct evidence by gene inactivation and/or heterologous expression that confirms the involvement of the cluster in pactamycin biosynthesis. Herein, we report the cloning and functional characterization of the pactamycin gene cluster in vivo by gene inactivation of the genes encoding the FeÀS radical SAM oxidoreductase (PtmC), the glycosyltransferase (PtmJ), and the polyketide synthase (PtmQ).…”

“…Based on BLAST similarity with the mitomycin biosynthetic genes and preliminary experiments, [11] it is predicted that PtmC, PtmJ (putative glycosyltransferase), and PtmG (putative deacetylase) might be involved in the formation of the cyclopentitol core 24 (Scheme 2), and that this process might be similar to the formation of the mitosane core structure during mitomycin (33) biosynthesis (Scheme 3). [17] Interestingly, the radical SAM enzyme MitD, the glycosyltransferase MitB, and the putative Ndeacetylase MitC from the mitomycin biosynthetic gene cluster are close homologues of PtmC, PtmJ, and PtmG, respectively.…”

“…[10] In order to further study the biosynthesis of pactamycin, we have identified the biosynthetic gene cluster of this important compound within an 86 kb sequenced region of DNA from S. pactum American Type Culture Collection (ATCC) 27456. More recently, Kudo, et al have independently reported the biosynthetic gene cluster of pactamycin from S. pactum NBRC 13 433, [11] which shares the same entry with the ATCC 27456 strain in the ATCC. The previously reported 34 kb DNA sequence consists of 24 open reading frames (ORFs) believed to be involved in the biosynthesis of pactamycin.…”

“…These include orf11 (encodes protein that shares high homology with the extracytoplasmic function (ECF) subfamily of RNA polymerase sigma factors), orf14 and orf15 (encode proteins that have low identity to translation initiation factor IF-2 from Frankia alni ACN4a and Streptomyces avermitilis MA-4680, respectively), orf9, orf16, and orf19 (encode proteins that are highly related to the family of ATP-dependent (DEAD-box) RNA helicases), orf18 (encodes protein that shares high homology with nourseothricin acetyltransferase from Streptomyces noursei), and orf23 (encodes protein that is highly similar to the tRNA methyltransferase from S. avermitilis). The pactamycin gene cluster reported by Kudo, et al consists of orf1, pctA-pctX, and orf2, À3, and À4, [11] which are identical to orf16, the ptm genes, and orf17 ( Table 1).…”

Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli

“…1–3 These enzymes methylate unactivated carbon and phosphorus centers, many of which are found in a growing number of natural products that display antibiotic or antineoplastic activity, as well as enzyme cofactors and other cellular metabolites, such as bacteriochlorophyll (enzyme cofactor), 4 pactamycin (antitumor agent and antibiotic), 5 mitomycin C (antitumor agent), 6 moenomycin A (phosphoglycolipid antibiotic), 7 fosfomycin (broad-spectrum antibiotic), 8–11 thienamycin (β-lactam antibiotic), 12 gentamicin (aminoglycoside antibiotic), 13 clorobiocin (aminocoumarin antibiotic), 14 fortimicin (aminoglycoside antibiotic), 15, 16 thiostrepton (thiopeptide antibiotic) 17 polytheonamide (secondary metabolite), 18, 19 quinomycin (peptide antibiotic), 20 and chondrochloren (antibiotic) 21 (Figure 1). They have been organized into four classes based on cofactor requirement, architecture, and mechanism of action.…”