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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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HPLC comparative analysis of the reactions catalysed by holo-module 5 and holo-M5*H790A with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)a. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) with holo-M5*H790A supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC comparison of unbound products from reaction mixtures containing L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) and either holo-module 5 or M5*H790A variant supplemented with ATP, L-pHPG, indicating the formation of β-lactam product only from the wild-type reaction. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H790A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. Pro-nocardicin G was not observed. (iii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (iv) HPLC trace of authentic standard of pro-nocardicin G.
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Figure 6: HPLC comparative analysis of the reactions catalysed by holo-module 5 and holo-M5*H790A with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)a. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) with holo-M5*H790A supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC comparison of unbound products from reaction mixtures containing L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) and either holo-module 5 or M5*H790A variant supplemented with ATP, L-pHPG, indicating the formation of β-lactam product only from the wild-type reaction. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H790A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. Pro-nocardicin G was not observed. (iii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (iv) HPLC trace of authentic standard of pro-nocardicin G.

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)

HPLC comparative analysis of the reactions catalysed by holo-module 5 and holo-M5*H790A with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)a. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) with holo-M5*H790A supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC comparison of unbound products from reaction mixtures containing L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) and either holo-module 5 or M5*H790A variant supplemented with ATP, L-pHPG, indicating the formation of β-lactam product only from the wild-type reaction. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H790A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. Pro-nocardicin G was not observed. (iii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (iv) HPLC trace of authentic standard of pro-nocardicin G.
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Related In: Results  -  Collection

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Figure 6: HPLC comparative analysis of the reactions catalysed by holo-module 5 and holo-M5*H790A with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2)a. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) with holo-M5*H790A supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC comparison of unbound products from reaction mixtures containing L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2) and either holo-module 5 or M5*H790A variant supplemented with ATP, L-pHPG, indicating the formation of β-lactam product only from the wild-type reaction. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H790A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. Pro-nocardicin G was not observed. (iii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP4 (2), L-pHPG and ATP. (iv) HPLC trace of authentic standard of pro-nocardicin G.
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