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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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Proposed β-lactam formation mechanisma, Proposed mechanism of β-lactam formation in C5. Tentative catalytic roles of histidine residues are indicated. b, Substrate used in this study. c, Incubation of dehydroalanyl tetrapeptidyl-S-PCP47 with holo-module 5, ATP and L-pHPG gave pro-nocardicin G. d, Left: HPLC traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). Right: LC-MS traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5 construct, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). TOF = time of flight, ES = electrospray ionization, EI = extracted ion.
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Figure 13: Proposed β-lactam formation mechanisma, Proposed mechanism of β-lactam formation in C5. Tentative catalytic roles of histidine residues are indicated. b, Substrate used in this study. c, Incubation of dehydroalanyl tetrapeptidyl-S-PCP47 with holo-module 5, ATP and L-pHPG gave pro-nocardicin G. d, Left: HPLC traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). Right: LC-MS traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5 construct, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). TOF = time of flight, ES = electrospray ionization, EI = extracted ion.

Mentions: Examination of the primary sequence of C5 showed no unusual insertions or deletions except that, in addition to the conserved HHxxxDG catalytic motif emblematic of condensation domains, a third His residue (H790) lies directly upstream of the His dyad (Extended Data Fig. 5). Analysis also revealed features of a DCL domain despite receiving an L-seryl tetrapeptide from PCP4 (Extended Data Table 1). We propose a mechanism in which His790 catalyses β-elimination of hydroxide (water) from the seryl residue of the PCP4-bound tetrapeptidyl-thioester and PCP5-tethered L-pHPG carries out β-addition with overall inversion of configuration at the seryl(dehydroalanyl) β-carbon dictated by earlier stereochemical experiments (Fig. 4a).25 The transient loss of the L-seryl stereocenter during the β-elimination/addition may account for the DCL characteristics of C5. The resulting β-aminothioester 6 is then proposed to undergo unconventional amide bond cyclisation (allowed 4-exo-trig), thermodynamically driven by amide bond formation from the active PCP4 thioester. The PCP5-bound pentapeptide β-lactam (pro-epi-nocardicin G) is poised for delivery to NocTE for C-terminal epimerisation and hydrolytic product release.


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

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

Proposed β-lactam formation mechanisma, Proposed mechanism of β-lactam formation in C5. Tentative catalytic roles of histidine residues are indicated. b, Substrate used in this study. c, Incubation of dehydroalanyl tetrapeptidyl-S-PCP47 with holo-module 5, ATP and L-pHPG gave pro-nocardicin G. d, Left: HPLC traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). Right: LC-MS traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5 construct, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). TOF = time of flight, ES = electrospray ionization, EI = extracted ion.
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Figure 13: Proposed β-lactam formation mechanisma, Proposed mechanism of β-lactam formation in C5. Tentative catalytic roles of histidine residues are indicated. b, Substrate used in this study. c, Incubation of dehydroalanyl tetrapeptidyl-S-PCP47 with holo-module 5, ATP and L-pHPG gave pro-nocardicin G. d, Left: HPLC traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). Right: LC-MS traces of products obtained after incubation of dehydroalanyl tetrapeptidyl-S-PCP47 and indicated holo-module 5 construct, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H790A), verified by comparison to synthetic standard (top trace). TOF = time of flight, ES = electrospray ionization, EI = extracted ion.
Mentions: Examination of the primary sequence of C5 showed no unusual insertions or deletions except that, in addition to the conserved HHxxxDG catalytic motif emblematic of condensation domains, a third His residue (H790) lies directly upstream of the His dyad (Extended Data Fig. 5). Analysis also revealed features of a DCL domain despite receiving an L-seryl tetrapeptide from PCP4 (Extended Data Table 1). We propose a mechanism in which His790 catalyses β-elimination of hydroxide (water) from the seryl residue of the PCP4-bound tetrapeptidyl-thioester and PCP5-tethered L-pHPG carries out β-addition with overall inversion of configuration at the seryl(dehydroalanyl) β-carbon dictated by earlier stereochemical experiments (Fig. 4a).25 The transient loss of the L-seryl stereocenter during the β-elimination/addition may account for the DCL characteristics of C5. The resulting β-aminothioester 6 is then proposed to undergo unconventional amide bond cyclisation (allowed 4-exo-trig), thermodynamically driven by amide bond formation from the active PCP4 thioester. The PCP5-bound pentapeptide β-lactam (pro-epi-nocardicin G) is poised for delivery to NocTE for C-terminal epimerisation and hydrolytic product release.

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