Nejvíce citovaný článek - PubMed ID 19917754
Mutasynthesis of lincomycin derivatives with activity against drug-resistant staphylococci
Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.
Lincosamides, which are produced by streptomycetes, compose a small but clinically important class of antibiotics. The recent elucidation of the condensation and post-condensation biosynthetic steps of the lincosamides lincomycin and celesticetin revealed several unexpected reaction mechanisms. Here, we prepared recombinant proteins involved in the celesticetin biosynthetic pathway and used them for in vitro assays that were monitored by LC-MS. Our results elucidate the last biosynthetic step of celesticetin: the attachment of salicylic acid is catalyzed by the Ccb2 acyl-CoA ligase and the Ccb1 acyltransferase. Ccb1 belongs to the WS/DGAT protein family and, in contrast to the characterized members of the family, has unusual substrate specificity. To the best of our knowledge, Ccb1 is the first protein in this family that transfers a benzoyl derivative-CoA conjugate and is the first WS/DGAT protein involved in the biosynthesis of secondary metabolites. Furthermore, we exploited the relaxed substrate specificities of Ccb1 and Ccb2, as well as three additional upstream post-condensation biosynthetic proteins in the celesticetin pathway, and combined the lincomycin and the celesticetin biosynthetic pathways in vitro. In this way, we prepared a library of 150 novel hybrid lincosamides, including two unnatural chimeras of lincomycin and celesticetin, which were shown to have antibacterial properties more pronounced than clinically used lincomycin. These achievements may be considered a case study in applying knowledge about biosynthetic machinery to assemble a large number of compounds from originally a small group of natural products without the need for chemical synthesis.
- Publikační typ
- časopisecké články MeSH
Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.
- Klíčová slova
- 4-propyl-L-proline, antibiotics, anticancer drug, hormaomycin, lincomycin, natural product biosynthesis, pyrrolobenzodiazepine, secondary metabolism,
- Publikační typ
- časopisecké články MeSH
In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.
- MeSH
- cystein metabolismus MeSH
- glykopeptidy metabolismus MeSH
- inositol metabolismus MeSH
- linkomycin biosyntéza MeSH
- linkosamidy biosyntéza MeSH
- peptidsynthasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- celesticetin A MeSH Prohlížeč
- cystein MeSH
- glykopeptidy MeSH
- inositol MeSH
- linkomycin MeSH
- linkosamidy MeSH
- mycothiol MeSH Prohlížeč
- peptidsynthasy MeSH
The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.
- MeSH
- anthramycin analogy a deriváty biosyntéza chemie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- molekulární struktura MeSH
- multigenová rodina * MeSH
- sekvenční analýza MeSH
- Streptomyces chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- anthramycin MeSH
- bakteriální proteiny MeSH
- porothramycin A MeSH Prohlížeč
Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accommodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin--but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit.
- MeSH
- bakteriální proteiny chemie MeSH
- dipeptidy chemie MeSH
- linkomycin biosyntéza chemie MeSH
- linkosamidy biosyntéza chemie MeSH
- molekulární evoluce * MeSH
- simulace molekulární dynamiky * MeSH
- Streptomyces enzymologie MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- celesticetin A MeSH Prohlížeč
- dipeptidy MeSH
- linkomycin MeSH
- linkosamidy MeSH
- prolyl-proline MeSH Prohlížeč
