Nejvíce citovaný článek - PubMed ID 10712928
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.
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
The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.
- MeSH
- fungální proteiny genetika MeSH
- klonování DNA metody MeSH
- peptidsynthasy genetika MeSH
- sekvenční analýza DNA MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Trichoderma enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fungální proteiny MeSH
- peptidsynthasy MeSH
