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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 spectrum of major product generated from reaction catalysed by holo-module 5 and L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) supplemented with ATP and L-pHPGThe major product from the reaction containing L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) was isolated over multiple injections by HPLC (peak indicated in inset HPLC trace) and then determined by HRMS (ESI+). The exact mass ion corresponding to the M+H ion of pro-nocardicin G was observed. Exact mass calculated for C33H39N8O9: 691.2835; Found: 691.2822 [M+H]+.
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Figure 8: Mass spectrum of major product generated from reaction catalysed by holo-module 5 and L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) supplemented with ATP and L-pHPGThe major product from the reaction containing L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) was isolated over multiple injections by HPLC (peak indicated in inset HPLC trace) and then determined by HRMS (ESI+). The exact mass ion corresponding to the M+H ion of pro-nocardicin G was observed. Exact mass calculated for C33H39N8O9: 691.2835; Found: 691.2822 [M+H]+.

Mentions: To support this mechanistic hypothesis, we prepared a mutant of module 5 in which the tentative catalytic His was replaced by alanine (M5*H790A). Repeating the experiments with PCP4-bound tetrapeptide 2 and mutant M5*H790A, L-pHPG and ATP yielded no product (Extended Data Fig. 6). Similarly site-specific mutation of the His residue typically involved in peptide bond formation (M5*H792A) also gave no reaction, as anticipated. In a further test of the proposed mechanism, the reactive dehydroalanyl tetrapeptide intermediate (6, Fig. 4b) was synthesised (Supplementary Information) and used in an Sfp-catalysed reaction to afford the corresponding L-pHPG–L-Arg–D-pHPG–dehydroalanyl-S-PCP4 substrate (7, Fig. 4c and Extended Data Fig. 7). In the course of preparing this sensitive material, it was discovered that the addition of sulfur, phosphorus and nitrogen nucleophiles occurred preferentially 1,4 rather than 1,2 in keeping with the hypothetical reactivity posed in Fig. 4a. When PCP4-bound dehydroalanyl tetrapeptide 7 was incubated with wild-type holo-module 5, L-pHPG and ATP, β-lactam formation was once again observed (Fig. 4d and Extended Data Fig. 8 and 9). Further insight into this process was afforded by the M5*H790A mutant, which did not support complete reaction of the dehydroalanyl substrate to the β-lactam product (Fig. 4d). The proposed catalytic residue H790 must not only act as a base to promote β-elimination but also serve as the acid to consummate amine (L-pHPG-S-PCP5) β-addition. Although interfering with the proper cycling of the protonation state of the enzyme can be partially compensated in the wild-type protein, it cannot in the M5*H790A mutant.


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

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

Mass spectrum of major product generated from reaction catalysed by holo-module 5 and L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) supplemented with ATP and L-pHPGThe major product from the reaction containing L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) was isolated over multiple injections by HPLC (peak indicated in inset HPLC trace) and then determined by HRMS (ESI+). The exact mass ion corresponding to the M+H ion of pro-nocardicin G was observed. Exact mass calculated for C33H39N8O9: 691.2835; Found: 691.2822 [M+H]+.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Mass spectrum of major product generated from reaction catalysed by holo-module 5 and L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) supplemented with ATP and L-pHPGThe major product from the reaction containing L-pHPG-L-Arg-D-pHPG-dehydroalanyl-S-PCP4 (7) was isolated over multiple injections by HPLC (peak indicated in inset HPLC trace) and then determined by HRMS (ESI+). The exact mass ion corresponding to the M+H ion of pro-nocardicin G was observed. Exact mass calculated for C33H39N8O9: 691.2835; Found: 691.2822 [M+H]+.
Mentions: To support this mechanistic hypothesis, we prepared a mutant of module 5 in which the tentative catalytic His was replaced by alanine (M5*H790A). Repeating the experiments with PCP4-bound tetrapeptide 2 and mutant M5*H790A, L-pHPG and ATP yielded no product (Extended Data Fig. 6). Similarly site-specific mutation of the His residue typically involved in peptide bond formation (M5*H792A) also gave no reaction, as anticipated. In a further test of the proposed mechanism, the reactive dehydroalanyl tetrapeptide intermediate (6, Fig. 4b) was synthesised (Supplementary Information) and used in an Sfp-catalysed reaction to afford the corresponding L-pHPG–L-Arg–D-pHPG–dehydroalanyl-S-PCP4 substrate (7, Fig. 4c and Extended Data Fig. 7). In the course of preparing this sensitive material, it was discovered that the addition of sulfur, phosphorus and nitrogen nucleophiles occurred preferentially 1,4 rather than 1,2 in keeping with the hypothetical reactivity posed in Fig. 4a. When PCP4-bound dehydroalanyl tetrapeptide 7 was incubated with wild-type holo-module 5, L-pHPG and ATP, β-lactam formation was once again observed (Fig. 4d and Extended Data Fig. 8 and 9). Further insight into this process was afforded by the M5*H790A mutant, which did not support complete reaction of the dehydroalanyl substrate to the β-lactam product (Fig. 4d). The proposed catalytic residue H790 must not only act as a base to promote β-elimination but also serve as the acid to consummate amine (L-pHPG-S-PCP5) β-addition. Although interfering with the proper cycling of the protonation state of the enzyme can be partially compensated in the wild-type protein, it cannot in the M5*H790A mutant.

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