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Structure and Function of a C–C Bond Cleaving Oxygenase in Atypical Angucycline Biosynthesis
C–C
bond ring cleaving oxygenases represent a unique family
of enzymes involved in the B ring cleavage reaction only observed
in atypical angucycline biosynthesis. B ring cleavage is the key reaction
leading to dramatic divergence in the final structures of atypical
angucyclines. Here, we present the crystal structure of AlpJ, the
first structure of this family of enzymes. AlpJ has been verified
as the enzyme catalyzing C–C bond cleavage in kinamycin biosynthesis.
The crystal structure of the AlpJ monomer resembles the dimeric structure
of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ
are homologous, and both contain a putative hydrophobic substrate
binding pocket in the “closed” and “open”
conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6
and protein–ligand docking analysis suggest that the residues
including Asn60, Trp64, and Trp181 are possibly involved in substrate
recognition. Site-directed mutagenesis results supported our hypothesis,
as mutation of these residues led to nearly a complete loss of the
activity of AlpJ. Structural analysis also revealed that AlpJ possesses
an intramolecular domain–domain interface, where the residues
His50 and Tyr178 form a hydrogen bond that probably stabilizes the
three-dimensional structure of AlpJ. Site-directed mutagenesis showed
that the two residues, His50 and Tyr178, were vital for the activity
of AlpJ. Our findings shed light on the structure and catalytic mechanism
of the AlpJ family of oxygenases, which presumably involves two active
sites that might function in a cooperative manner