Monobactam formation in sulfazecin by a nonribosomal peptide synthetase thioesterase.

The N-sulfonated monocyclic ß-lactam ring characteristic of the monobactams confers resistance to zinc metallo-ß-lactamases and affords the most effective class to combat carbapenem-resistant enterobacteria (CRE). Here we report unprecedented nonribosomal peptide synthetase activities, wherein an assembled tripeptide is N-sulfonated in trans before direct synthesis of the ß-lactam ring in a noncanonical, cysteine-containing thioesterase domain. This means of azetidinone synthesis is distinct from the three others known in nature.

Biosynthesis of active pharmaceuticals: ß-lactam biosynthesis in filamentous fungi.

ß-lactam antibiotics (e.g. penicillins, cephalosporins) are of major clinical importance and contribute to over 40% of the total antibiotic market. These compounds are produced as secondary metabolites by certain actinomycetes and filamentous fungi (e.g. Penicillium, Aspergillus and Acremonium species). The industrial producer of penicillin is the fungus Penicillium chrysogenum. The enzymes of the penicillin biosynthetic pathway are well characterized and most of them are encoded by genes that are organized in a cluster in the genome. Remarkably, the penicillin biosynthetic pathway is compartmentalized: the initial steps of penicillin biosynthesis are catalyzed by cytosolic enzymes, whereas the two final steps involve peroxisomal enzymes. Here, we describe the biochemical properties of the enzymes of ß-lactam biosynthesis in P. chrysogenum and the role of peroxisomes in this process. An overview is given on strain improvement programs via classical mutagenesis and, more recently, genetic engineering, leading to more productive strains. Also, the potential of using heterologous hosts for the development of novel ß-lactam antibiotics and non-ribosomal peptide synthetase-based peptides is discussed.

One Ring to Fight Them All: The Sulfazecin Story.

In this issue of Cell Chemical Biology, Li et al. (2017) report on the biosynthesis of the monobactam sulfazecin by Pseudomonas acidophila and hypothesize a novel mechanism of ß-lactam ring formation. As monobactam antibiotics are unaffected by some emerging resistance mechanisms (particularly metallo-ß-lactamases), this discovery opens prospects to engineer ß-lactam antibiotics against multi-drug resistant pathogens.

Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster.

The monobactams, exemplified by the natural product sulfazecin, are the only class of ß-lactam antibiotics not inactivated by metallo-ß-lactamases, which confer bacteria with extended-spectrum ß-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the ß-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and ß-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic ß-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.

Measurement of the degree of coupled isotopic enrichment of different positions in an antibiotic peptide by NMR.

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coupled site. They supplement existing methods for determining the extent of isotopic incorporation at individual sites to reveal whether two coupled labeled sites in a precursor are incorporated as an intact unit into products. The methods described also circumvent 1H signal overlap and distinguish between the effects of different nitrogens coupled to individual carbons. The somewhat complicated case of valclavam illustrates the method's utility in measuring the J coupling constants between 13C and nearby sites that are only fractionally labeled with 15N, and measuring the fraction of molecules in which 13C is coupled to 15N, at each of several sites. The 15N of [2-13C, 15N]-labeled glycine is found to be incorporated into all three N positions of valclavam but most heavily into the N11 position. Specifically, 15N and 13C are incorporated into the N11 and C10 positions together as an 15N13C fragment approximately 8% of the time, whereas 15N is incorporated largely independently at the other positions.

MM 42842, a new member of the monobactam family produced by Pseudomonas cocovenenans. I. Identification of the producing organism.

A bacterial soil isolate designated 326-32B produces a new member of the monobactam series of antibiotics, MM 42842, and the bulgecins. Identification studies show isolate 326-32B to be a strain of Pseudomonas cocoveneans which is a species previously noted for the production of toxoflavin. A description of P. cocovenenans does not appear to have been previously published and the identify of strain 326-32B was established by means of a direct comparison with the deposited organism P. cocovenenans NCIB 9450. The properties of strain 326-32B, and P. cocovenenans NCIB 9450 were compared with those of the monobactam and bulgecin producing organisms Pseudomonas acidophila ATCC 31363 and Pseudomonas mesoacidophila ATCC 31433. The four organisms were found to share certain properties, including the ability to grow at pH 4.0.

Enzymatic synthesis of monocyclic beta-lactams.

An Mg2+ and ATP dependent beta-lactam synthetase (BLS) catalyses formation of a beta-lactam ring during the biosynthesis of clavulanic acid, an important beta-lactamase inhibitor. An epimeric mixture of a 2-methylated derivative of the natural BLS substrate N2-(2-carboxyethyl)-L-arginine was synthesised and found to be a substrate for the enzyme. The epimeric products were characterised by 1H NMR and mass spectrometric analyses. The results suggest that a modified version of BLS might be used to catalyse the preparation of intermediates useful for the synthesis of beta-lactam antibiotics.

Influence of growth rate and nutrient limitation on monobactam production and peptidoglycan synthesis in Pseudomonas aeruginosa.

The effects of growth rate and nutrient limitation on monobactam production, peptidoglycan content and mean cell length in Pseudomonas aeruginosa was studied using continuous culture techniques. All three parameters increased progressively with growth rate, a greater response being shown under carbon limitation compared to that occurring under nitrogen limiting conditions. Interestingly, monobactum production mirrored peptidoglycan synthesis. In addition, the monobactam exhibited a broad range of antibacterial activity and bound preferentially to PBP 1A in the producing organism. Moreover, addition of the monobactam to a growing culture inhibited cell wall synthesis. These results are discussed in relation to the control and regulation of peptidoglycan synthesis.