title: Adaptation of an L-proline adenylation domain to use 4-propyl-L-proline in the evolution of lincosamide biosynthesis.
abstract: 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.
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Kadlčík
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kučera
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Dominika
authors.initials: D
authors.lastname: Chalupská
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Gažák
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Markéta
authors.initials: M
authors.lastname: Koběrská
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Ulanová
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Kopecký
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic ; Department of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovac Academy of Sciences, Bratislava, Slovakia.
authors.forename: Eva
authors.initials: E
authors.lastname: Kutejová
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Najmanová
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Janata
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry*; D019143:Evolution, Molecular; D008034:Lincomycin / Q000096:biosynthesis* / Q000737:chemistry; D055231:Lincosamides / Q000096:biosynthesis* / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D055231:Lincosamides; C448039:prolyl-proline; C004650:celesticetin A; D008034:Lincomycin