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Kudo F,
Kasama Y,
Hirayama T,
Eguchi T,
( 2007 ) Cloning of the pactamycin biosynthetic gene cluster and characterization of a crucial glycosyltransferase prior to a unique cyclopentane ring formation. PMID : 17827660 : DOI : 10.1038/ja.2007.63 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-alpha-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 beta-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.
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2. |
Hirayama A,
Miyanaga A,
Kudo F,
Eguchi T,
( 2015 ) Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin. PMID : 26426567 : DOI : 10.1002/cbic.201500426 Abstract >>
Mutational analysis of the pyridoxal 5'-phosphate (PLP)-dependent enzyme PctV was carried out to elucidate the multi-step reaction mechanism for the formation of 3-aminobenzoate (3-ABA) from 3-dehydroshikimate (3-DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5'-phosphate (PMP) with 3-DSA. The chemical structure of this intermediate was supported by X-ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3-ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.
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3. |
Chen R,
Zhang Q,
Tan B,
Zheng L,
Li H,
Zhu Y,
Zhang C,
( 2017 ) Genome Mining and Activation of a Silent PKS/NRPS Gene Cluster Direct the Production of Totopotensamides. PMID : 29019409 : DOI : 10.1021/acs.orglett.7b02878 Abstract >>
A 92 kb silent hybrid polyketide and nonribosomal peptide gene cluster in marine-derived Streptomyces pactum SCSIO 02999 was activated by genetically manipulating the regulatory genes, including the knockout of two negative regulators (totR5 and totR3) and overexpression of a positive regulator totR1, to direct the production of the known totopotensamides (TPMs) A (1) and B (3) and a novel sulfonate-containing analogue TPM C (2). Inactivation of totG led to accumulation of TPM B (3) lacking the glycosyl moiety, which indicated TotG as a dedicated glycosyltransferase in the biosynthesis of 1 and 2.
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