Origin of the β-Lactam Carbons in Clavulanic Acid from an Unusual Thiamine Pyrophosphate-Mediated Reaction

Khaleeli, Nusrat; Li, Rongfeng; Townsend, Craig A.

doi:10.1021/ja9923134

Cited by 90 publications

(85 citation statements)

References 20 publications

(46 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…NocJ-catalyzed Deuterium Exchange in Nocardicin A-NocJ enzyme buffer was exchanged with deuterated enzyme buffer (50 mM sodium phosphate, 10% glycerol-OD 3 Kinetic Measurements of NocJ with Isonocardicin A and Nocardicin A-Calibration curves of both isonocardicin A and nocardicin A derivatized with GITC were prepared using 1,3,5-trimethoxybenzene as an internal standard. Assay mixtures were prepared with set concentrations of N-His 6 -NocJ (0.02 M and 0.04 M for isonocardicin A; 0.08 mM for nocardicin A) in 100 l, (1 mM and higher of substrate) 200 l (0.5 mM substrate), 500 l (0.1 mM substrate), or 1000 l (0.05 mM substrate) and 40 M PLP in 50 mM sodium phosphate, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…T he characterization of natural product biosynthetic pathways at the genetic level has revealed a growing number of examples in which the synthetic capability of a class of enzymes has been directed to a different end (1,2). A particularly striking instance is the relationship between Streptomyces clavuligerus ␤-lactam synthetase (␤-LS) and the universally distributed and highly conserved family of asparagine synthetases, class B (AS-Bs).…”

mentioning

confidence: 99%

The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

Miller

Bachmann

Townsend

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The catalytic cycle of the ATP͞Mg 2؉ -dependent enzyme ␤-lactam synthetase (␤-LS) from Streptomyces clavuligerus has been observed through a series of x-ray crystallographic snapshots. Chemistry is initiated by the ordered binding of ATP͞Mg 2؉ and N 2 -(carboxyethyl)-L-arginine (CEA) to the apoenzyme. The apo and ATP͞Mg 2؉ structures described here, along with the previously described CEA⅐␣,␤-methyleneadenosine 5 -triphosphate (CEA⅐AMP-CPP)͞Mg 2؉ structure, illuminate changes in active site geometry that favor adenylation. In addition, an acyladenylate intermediate has been trapped. The substrate analog N 2 -(carboxymethyl)-L-arginine (CMA) was adenylated by ATP in the crystal and represents a close structural analog of the previously proposed CEA-adenylate intermediate. Finally, the structure of the ternary product complex deoxyguanidinoproclavaminic acid (DGPC)⅐AMP͞PP i͞Mg 2؉ has been determined. The CMA-AMP͞PP i͞Mg 2؉ and DGPC⅐AMP͞PPi͞Mg 2؉ structures reveal interactions in the active site that facilitate ␤-lactam formation. All of the ATP-bound structures differ from the previously described CEA⅐AMP-CPP͞Mg 2؉ structure in that two Mg 2؉ ions are found in the active sites. These Mg 2؉ ions play critical roles in both the adenylation and ␤-lactamization reactions.

show abstract

“…It cannot be excluded that product 6 is an artefact of the column chromatography used trying to isolate possible addition products. However, Townsend and coworkers [66] report that the hydrolysis of a 2-acylated NHC leads to the formation of the corresponding carboxylic acid; indeed we evidenced the formation of phenylacetic acid in this experiment, but we cannot exclude a simple hydrolysis of acyl chloride during the workup. From these results, it seems difficult to understand the mechanism of this Staudinger synthesis.…”

Section: Resultsmentioning

confidence: 44%

Investigation of the Role of Electrogenerated N-Heterocyclic Carbene in the Staudinger Synthesis in Ionic Liquid

Feroci¹

2011

IJOC

View full text Add to dashboard Cite

Electrogenerated N-heterocyclic carbene (NHC), obtained by cathodic reduction of Bmim-BF 4 , behaves as organocatalyst and base in the Staudinger synthesis from an acyl chloride and a deactivated imine in ionic liquid to yield -lactams. The effect of many parameters (temperature, amount of electricity, substituents, presence of an external base) has been evaluated and a tentative mechanism for the Staudinger synthesis in a very polar medium like the ionic liquid reported. The yields of isolated -lactams are good, starting from non-electrophilic imines, and predominantly trans lactams are obtained with a good diastereomeric ratio.

show abstract

Origin of the β-Lactam Carbons in Clavulanic Acid from an Unusual Thiamine Pyrophosphate-Mediated Reaction

Cited by 90 publications

References 20 publications

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

The catalytic cycle of β-lactam synthetase observed by x-ray crystallographic snapshots

Investigation of the Role of Electrogenerated N-Heterocyclic Carbene in the Staudinger Synthesis in Ionic Liquid

Contact Info

Product

Resources

About