Cell-free biosynthesis of nocardicin A from nocardicin E and S-adenosylmethionine

Wilson, Brenda A.; Bantia, Shanta; Salituro, Gino; Reeve, Anne M.; Townsend, Craig A.

doi:10.1021/ja00232a047

Cited by 18 publications

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“…However, if a peak of this magnitude did corre- spond to nocardicin A, at least some degree of antibiosis should have been plainly visible in the E. coli SC12155 bioassay. Nocardicin A and isonocardicin A are indistinguishable under typical reverse phase HPLC conditions, (46) and examination by chiral HPLC did confirm the absence of nocardicin A. Isolation of the compound and further characterization established its identity as isonocardicin A.…”

Section: Analysis Of Nocj-mentioning

confidence: 90%

“…Initial in vitro assays by HPLC and mass spectrometric analysis of NocJ confirmed its role as the C-9Ј epimerase active with both isonocardicin A and isonocardicin C, as well as the reverse reaction from nocardicin A and nocardicin C. It is a limitation that isonocardicin A and nocardicin A are indistinguishable by reverse phase HPLC, UV-visible spectroscopy, mass spectrometry, and 400 MHz 1 H NMR (46). To overcome this obstacle, and to show unambiguously the epimerization at C-9Ј of nocardicin A, the reaction was carried out in deuterated buffer.…”

Section: Analysis Of Nocj-mentioning

confidence: 95%

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Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

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The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic ␤-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a D-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5 -phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the L-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9 epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of ␤-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.

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Section: Analysis Of Nocj-mentioning

confidence: 90%

Section: Analysis Of Nocj-mentioning

confidence: 95%

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

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“…Properties-The product of NAT was expected to be isonocardicin A (L at C-9Ј), resulting from direct S N 2 displacement of the 3-amino-3-carboxypropyl group of L-AdoMet by the phenolic hydroxyl group of nocardicin E. An authentic sample of isonocardicin A was found to be indistinguishable from nocardicin A by the HPLC conditions of the standard assay as well as by UV and 1 H NMR spectroscopy at 400 MHz (19). The presence of an epimerase capable of interconverting nocardicin A and isonocardicin A has been demonstrated in crude and partially purified cell-free extracts of N. uniformis (19); therefore, the actual stereochemical requirements at C-9Ј of the transferase reaction remained in doubt.…”

Section: Resultsmentioning

confidence: 99%

“…This configurational outcome parallels the stereochemistry of addition of the 3-aminopropyl moiety derived from decarboxylated S-adenosylmethionine (AdoMet) 1 operative in the biosynthesis of the polyamines (14 -16), as well as more conventional methyltransferases (17,18), suggesting a role for AdoMet in nocardicin A biosynthesis in vivo. This postulate was borne out in preliminary cell-free studies in which an efficient, time-dependent conversion from nocardicin E (5) to nocardicin A in the presence of AdoMet was demonstrated (19). Assuming the epimerization of the amino acid terminus (C-9Ј) of nocardicin A is a late step in the biosynthesis, the product of this transferase enzyme was expected to be isonocardicin A (L at C-9Ј).…”

mentioning

confidence: 99%

“…Assuming the epimerization of the amino acid terminus (C-9Ј) of nocardicin A is a late step in the biosynthesis, the product of this transferase enzyme was expected to be isonocardicin A (L at C-9Ј). Subsequently, it was demonstrated that an epimerase capable of equilibrating the stereochemistry at C-9Ј of nocardicin A was also present in the cell-free extract (19).…”

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Purification, Characterization, and Cloning of an S-Adenosylmethionine-dependent 3-Amino-3-carboxypropyltransferase in Nocardicin Biosynthesis

Reeve

Breazeale

Townsend

1998

Journal of Biological Chemistry

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S-Adenosylmethionine:nocardicin 3-amino-3-carboxypropyltransferase catalyzes the biosynthetically rare transfer of the 3-amino-3-carboxypropyl moiety from S-adenosylmethionine to a phenolic site in the ␤-lactam substrates nocardicin E, F, and G, a late step of the biosynthesis of the monocyclic ␤-lactam antibiotic nocardicin A. Whereas a number of conventional methods were ineffective in purifying the transferase, it was successfully obtained by two complementary affinity chromatography steps that took advantage of the two substrate-two product reaction scheme. S-Adenosylhomocysteine-agarose selected enzymes that utilize S-adenosylmethionine, and a second column, nocardicin Aagarose, specifically bound the desired transferase to yield the enzyme as a single band of 38 kDa on a silverstained SDS-polyacrylamide gel. The transferase is active as a monomer and exhibits sequential kinetics. Further kinetic characterization of this protein is described and its role in the biosynthesis of nocardicin A discussed. The gene encoding this transferase was cloned from a sublibrary of Nocardia uniformis DNA. Translation gave a protein of deduced mass 32,386 Da which showed weak homology to small molecule methyltransferases. However, three correctly disposed signature motifs characteristic of these enzymes were observed.

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Four‐Membered Heterocyclic Rings and Their Fused Derivatives

2015

Biosynthesis of Heterocycles

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Cell-free biosynthesis of nocardicin A from nocardicin E and S-adenosylmethionine

Cited by 18 publications

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Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Purification, Characterization, and Cloning of an S-Adenosylmethionine-dependent 3-Amino-3-carboxypropyltransferase in Nocardicin Biosynthesis

Four‐Membered Heterocyclic Rings and Their Fused Derivatives

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