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β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis.

Gaudelli NM, Long DH, Townsend CA - Nature (2015)

Bottom Line: Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase).We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems.These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA.

ABSTRACT
Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

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Mass spectrometric verification of apo to holo conversion of PCP4 with Sfp and L-pHPG-L-Arg-D-pHPG-L-Ser-S-coenzyme A (1), forming L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic tetrapeptidyl-CoA substrate 1.
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Figure 1: Mass spectrometric verification of apo to holo conversion of PCP4 with Sfp and L-pHPG-L-Arg-D-pHPG-L-Ser-S-coenzyme A (1), forming L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic tetrapeptidyl-CoA substrate 1.

Mentions: The termination module of NocB, module 5, is composed of four domains: C5, A5, PCP5 and TE. This 144 kDa protein was heterologously expressed in Escherichia coli with a His6 tag and purified by affinity chromatography. Complete conversion to its corresponding holo form was ensured by Sfp-mediated 4′-phosphopantetheinyl transfer from coenzyme A (CoASH).22,23 The final chemical transformations catalysed by the termination module were successfully reconstituted in vitro through incubation of the predicted tetrapeptide-modified PCP domain from module 4 (PCP4) with holo-module 5. Bearing in mind that all five modules of NocA/B are required for production of nocardicin A in N. uniformis, and that the β-lactam-containing pentapeptide is preferentially processed by NocTE over the corresponding tripeptide,18 L-pHPG–L-Arg–D-pHPG–L-Ser-CoA (1, Fig 3a) was prepared (Supplementary Information) and linked to apo-PCP4 in an Sfp-mediated transfer to create L-pHPG–L-Arg–D-pHPG–L-Ser-S-PCP4 (2, Fig. 3b and Extended Data Fig. 1). It was anticipated that module 5 would activate L-pHPG in the presence of ATP and present this amino acid on PCP5 for reaction with the tetrapeptide delivered to module 5 by PCP4. Indeed, when holo-module 5, 10 equivalents of tetrapeptidyl-S-PCP42, L-pHPG and ATP were combined, smooth conversion to the pentapeptide β-lactam (pro-nocardicin G) was observed (Fig. 3c and Extended Data Fig. 2). Monitoring product formation by HPLC in a time-course experiment and simultaneous consumption of tetrapeptidyl-S-PCP42 by ESI-MS revealed a 1:1 correlation in accord with full catalytic turnover (Fig. 3d). Control experiments lacking L-pHPG or L-pHPG and ATP showed no product formation.


β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis.

Gaudelli NM, Long DH, Townsend CA - Nature (2015)

Mass spectrometric verification of apo to holo conversion of PCP4 with Sfp and L-pHPG-L-Arg-D-pHPG-L-Ser-S-coenzyme A (1), forming L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic tetrapeptidyl-CoA substrate 1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401618&req=5

Figure 1: Mass spectrometric verification of apo to holo conversion of PCP4 with Sfp and L-pHPG-L-Arg-D-pHPG-L-Ser-S-coenzyme A (1), forming L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)Top: mass spectrum (ESI+) of apo-PCP4. Bottom: mass spectrum (ESI+) of L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) derived from treatment of apo-PCP4 with Sfp and corresponding synthetic tetrapeptidyl-CoA substrate 1.
Mentions: The termination module of NocB, module 5, is composed of four domains: C5, A5, PCP5 and TE. This 144 kDa protein was heterologously expressed in Escherichia coli with a His6 tag and purified by affinity chromatography. Complete conversion to its corresponding holo form was ensured by Sfp-mediated 4′-phosphopantetheinyl transfer from coenzyme A (CoASH).22,23 The final chemical transformations catalysed by the termination module were successfully reconstituted in vitro through incubation of the predicted tetrapeptide-modified PCP domain from module 4 (PCP4) with holo-module 5. Bearing in mind that all five modules of NocA/B are required for production of nocardicin A in N. uniformis, and that the β-lactam-containing pentapeptide is preferentially processed by NocTE over the corresponding tripeptide,18 L-pHPG–L-Arg–D-pHPG–L-Ser-CoA (1, Fig 3a) was prepared (Supplementary Information) and linked to apo-PCP4 in an Sfp-mediated transfer to create L-pHPG–L-Arg–D-pHPG–L-Ser-S-PCP4 (2, Fig. 3b and Extended Data Fig. 1). It was anticipated that module 5 would activate L-pHPG in the presence of ATP and present this amino acid on PCP5 for reaction with the tetrapeptide delivered to module 5 by PCP4. Indeed, when holo-module 5, 10 equivalents of tetrapeptidyl-S-PCP42, L-pHPG and ATP were combined, smooth conversion to the pentapeptide β-lactam (pro-nocardicin G) was observed (Fig. 3c and Extended Data Fig. 2). Monitoring product formation by HPLC in a time-course experiment and simultaneous consumption of tetrapeptidyl-S-PCP42 by ESI-MS revealed a 1:1 correlation in accord with full catalytic turnover (Fig. 3d). Control experiments lacking L-pHPG or L-pHPG and ATP showed no product formation.

Bottom Line: Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase).We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems.These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA.

ABSTRACT
Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

Show MeSH
Related in: MedlinePlus