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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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Negative experiments resulting from incubation of holo-module 5 with alternate substratesa. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) with holo-module 5 supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC traces of products obtained after incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. Pro-nocardicin G was not observed as verified by comparison to authentic standard. Additionally, it was noted that substrate 3 was not consumed over the duration of the experiment. (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-pantetheine (3), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP. (iii) HPLC trace of incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP without enzyme. The peak corresponding to L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) substrate is indicated. (iv) HPLC trace of authentic standard of pro-nocardicin G. c. Schematic of incubation of the dipeptidyl substrate D-pHPG-L-Ser-S-PCP4 (5) with holo-module 5, L-pHPG and ATP. Nocardicin G, the corresponding expected product, was not observed. d. LC-MS traces of products obtained after incubation of D-pHPG-L-Ser-S-PCP4 (5) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. The corresponding β-lactam product was not observed as verified by comparison to authentic standard. (i) Total ion chromatogram of the unbound products resulting from wild-type holo-module 5 reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP (ii) Extracted ion chromatogram of the wild-type reaction in trace i, the 386.1 m/z ion, corresponding to [M+H] of nocardicin G was not observed. (iii) Total ion chromatogram of unbound products from holo-M5*H792A control reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP. (iv) Extracted ion chromatogram of the wild-type reaction in trace iii. (v) Extracted ion chromatogram of authentic standard of nocardicin G.
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Figure 3: Negative experiments resulting from incubation of holo-module 5 with alternate substratesa. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) with holo-module 5 supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC traces of products obtained after incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. Pro-nocardicin G was not observed as verified by comparison to authentic standard. Additionally, it was noted that substrate 3 was not consumed over the duration of the experiment. (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-pantetheine (3), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP. (iii) HPLC trace of incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP without enzyme. The peak corresponding to L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) substrate is indicated. (iv) HPLC trace of authentic standard of pro-nocardicin G. c. Schematic of incubation of the dipeptidyl substrate D-pHPG-L-Ser-S-PCP4 (5) with holo-module 5, L-pHPG and ATP. Nocardicin G, the corresponding expected product, was not observed. d. LC-MS traces of products obtained after incubation of D-pHPG-L-Ser-S-PCP4 (5) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. The corresponding β-lactam product was not observed as verified by comparison to authentic standard. (i) Total ion chromatogram of the unbound products resulting from wild-type holo-module 5 reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP (ii) Extracted ion chromatogram of the wild-type reaction in trace i, the 386.1 m/z ion, corresponding to [M+H] of nocardicin G was not observed. (iii) Total ion chromatogram of unbound products from holo-M5*H792A control reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP. (iv) Extracted ion chromatogram of the wild-type reaction in trace iii. (v) Extracted ion chromatogram of authentic standard of nocardicin G.

Mentions: In a negative control experiment, the in vitro reconstitution experiment was repeated with a point mutant of C5 where the second histidine residue of the conserved active site HHxxxDG sequence, known to be essential for amide bond formation,24 was replaced by alanine (H792A). No new products were detected (Fig. 3c). To further define acceptable substrates for C5, the L-pHPG–L-Arg–D-pHPG–L-Ser-S-pantetheine (3, Fig 3a) substrate mimic was prepared (Supplementary Information) but did not yield pro-nocardicin G when incubated with holo-module 5, ATP and L-pHPG (Extended Data Fig. 3a and b), a result that underscores the critical importance PCP4•C5 domain•domain interaction plays to β-lactam formation. Next the dipeptide D-pHPG–L-Ser-CoA (4, Fig 3a) was prepared (Supplementary Information) and loaded onto apo-PCP4 as before to afford D-pHPG–L-Ser-S-PCP4 (5, Extended Data Fig. 4). When this construct was generated in the presence of holo-module 5, L-pHPG and ATP, nocardicin G was not detected (Extended Data Fig. 3c and d). These data suggest that the L-pHPG–L-Arg “leader” present in the tetrapeptidyl-S-PCP42 plays a vital role in the binding and/or recognition of the upstream tetrapeptidyl intermediate in C5, enabling peptide extension and β-lactam formation to occur.


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

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

Negative experiments resulting from incubation of holo-module 5 with alternate substratesa. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) with holo-module 5 supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC traces of products obtained after incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. Pro-nocardicin G was not observed as verified by comparison to authentic standard. Additionally, it was noted that substrate 3 was not consumed over the duration of the experiment. (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-pantetheine (3), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP. (iii) HPLC trace of incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP without enzyme. The peak corresponding to L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) substrate is indicated. (iv) HPLC trace of authentic standard of pro-nocardicin G. c. Schematic of incubation of the dipeptidyl substrate D-pHPG-L-Ser-S-PCP4 (5) with holo-module 5, L-pHPG and ATP. Nocardicin G, the corresponding expected product, was not observed. d. LC-MS traces of products obtained after incubation of D-pHPG-L-Ser-S-PCP4 (5) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. The corresponding β-lactam product was not observed as verified by comparison to authentic standard. (i) Total ion chromatogram of the unbound products resulting from wild-type holo-module 5 reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP (ii) Extracted ion chromatogram of the wild-type reaction in trace i, the 386.1 m/z ion, corresponding to [M+H] of nocardicin G was not observed. (iii) Total ion chromatogram of unbound products from holo-M5*H792A control reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP. (iv) Extracted ion chromatogram of the wild-type reaction in trace iii. (v) Extracted ion chromatogram of authentic standard of nocardicin G.
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Figure 3: Negative experiments resulting from incubation of holo-module 5 with alternate substratesa. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) with holo-module 5 supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC traces of products obtained after incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. Pro-nocardicin G was not observed as verified by comparison to authentic standard. Additionally, it was noted that substrate 3 was not consumed over the duration of the experiment. (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-pantetheine (3), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP. (iii) HPLC trace of incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP without enzyme. The peak corresponding to L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) substrate is indicated. (iv) HPLC trace of authentic standard of pro-nocardicin G. c. Schematic of incubation of the dipeptidyl substrate D-pHPG-L-Ser-S-PCP4 (5) with holo-module 5, L-pHPG and ATP. Nocardicin G, the corresponding expected product, was not observed. d. LC-MS traces of products obtained after incubation of D-pHPG-L-Ser-S-PCP4 (5) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. The corresponding β-lactam product was not observed as verified by comparison to authentic standard. (i) Total ion chromatogram of the unbound products resulting from wild-type holo-module 5 reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP (ii) Extracted ion chromatogram of the wild-type reaction in trace i, the 386.1 m/z ion, corresponding to [M+H] of nocardicin G was not observed. (iii) Total ion chromatogram of unbound products from holo-M5*H792A control reaction with D-pHPG-L-Ser-S-PCP4 (5), L-pHPG and ATP. (iv) Extracted ion chromatogram of the wild-type reaction in trace iii. (v) Extracted ion chromatogram of authentic standard of nocardicin G.
Mentions: In a negative control experiment, the in vitro reconstitution experiment was repeated with a point mutant of C5 where the second histidine residue of the conserved active site HHxxxDG sequence, known to be essential for amide bond formation,24 was replaced by alanine (H792A). No new products were detected (Fig. 3c). To further define acceptable substrates for C5, the L-pHPG–L-Arg–D-pHPG–L-Ser-S-pantetheine (3, Fig 3a) substrate mimic was prepared (Supplementary Information) but did not yield pro-nocardicin G when incubated with holo-module 5, ATP and L-pHPG (Extended Data Fig. 3a and b), a result that underscores the critical importance PCP4•C5 domain•domain interaction plays to β-lactam formation. Next the dipeptide D-pHPG–L-Ser-CoA (4, Fig 3a) was prepared (Supplementary Information) and loaded onto apo-PCP4 as before to afford D-pHPG–L-Ser-S-PCP4 (5, Extended Data Fig. 4). When this construct was generated in the presence of holo-module 5, L-pHPG and ATP, nocardicin G was not detected (Extended Data Fig. 3c and d). These data suggest that the L-pHPG–L-Arg “leader” present in the tetrapeptidyl-S-PCP42 plays a vital role in the binding and/or recognition of the upstream tetrapeptidyl intermediate in C5, enabling peptide extension and β-lactam formation to occur.

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