Mechanistic and Structural Basis of Stereospecific Cβ-Hydroxylation in Calcium-Dependent Antibiotic, a Daptomycin-Type Lipopeptide
2007; American Chemical Society; Volume: 2; Issue: 3 Linguagem: Inglês
10.1021/cb700012y
ISSN1554-8937
AutoresMatthias Strieker, Florian Kopp, Christoph Mahlert, Lars‐Oliver Essen, Mohamed A. Marahiel,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoNon-ribosomally synthesized lipopeptide antibiotics of the daptomycin type are known to contain unnatural β-modified amino acids, which are essential for bioactivity. Here we present the biochemical and structural basis for the incorporation of 3-hydroxyasparagine at position 9 in the 11-residue acidic lipopeptide lactone calcium-dependent antibiotic (CDA). Direct hydroxylation of l-asparagine by AsnO, a non-heme Fe2+/α-ketoglutarate-dependent oxygenase encoded by the CDA biosynthesis gene cluster, was validated by Fmoc derivatization of the reaction product and LC/MS analysis. The 1.45, 1.92, and 1.66 Å crystal structures of AsnO as apoprotein, Fe2+ complex, and product complex, respectively, with (2S,3S)-3-hydroxyasparagine and succinate revealed the stereoselectivity and substrate specificity of AsnO. The comparison of native and product-complex structures of AsnO showed a lid-like region (residues F208–E223) that seals the active site upon substrate binding and shields it from sterically demanding peptide substrates. Accordingly, β-hydroxylated asparagine is synthesized prior to its incorporation into the growing CDA peptide. The AsnO structure could serve as a template for engineering novel enzymes for the synthesis of β-hydroxylated amino acids.
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