Journal:Acta Cryst F:S2053230X19002863
Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesisJolanta Cieślak, Akimasa Miyanaga, Makoto Takaishi, Fumitaka Kudo, Tadashi Eguchi [1] Molecular Tour This paper describes the biochemical and structural analyses of adenylation enzyme IdnL7 involved in the biosynthesis of macrolactam antibiotic incednine. IdnL7 shows a broad substrate specificity for several small L-amino acids such as L-alanine and glycine. To obtain mechanistic insights into the substrate recognition of IdnL7, we determined the crystal structure of IdnL7 in complex with a reaction intermediate analog. IdnL7 has Cys217, Ala285 and Thr318 at the substrate binding pocket. These residues likely enable to accommodate various small L-amino acids as a substrate. This structural observation expands our understanding of the structure-function relationships of adenylation enzymes. Cartoon representation of the overall structure of IdnL7 (PDB entry 6akd). The overall structure of IdnL7 consists of two domains: a large N-terminal domain (Met1–Gly413; in pink) and a smaller C-terminal domain (Gln420–Leu522; in magenta). Both domains are connected by a flexible hinge region (Arg414–Leu419), which allows for the rotation of the C-terminal domain during the two catalytic reaction steps. The N-terminal domain forms a five-layered αβαβα-sandwich fold, whereas the C-terminal domain comprises three helices with one two-stranded and one three-stranded antiparallel β-sheet. The C-terminal domain is arranged in the adenylation conformation in relation to the N-terminal domain. Ligand binding site. The L-Ala-SA molecule and residues involved in interactions with the ligand are shown as cyan and pink ball-and-sticks, respectively. Red balls represent the positions of water molecules. White dashed lines indicate hydrogen bonding. The adenine moiety is buried in a hydrophobic pocket that is lined by Tyr310, Tyr411 and Ile340 on one side, and a loop (residues Gly286–Ala288) on the other side. This architecture stabilizes the purine base by hydrophobic and van der Waals interactions. Additionally, the N6 amino group of the adenine ring interacts with the main chain carbonyl of Leu309 and the side-chain amide group of Asn308. Several water molecules participate in direct interactions with the sulfamoyladenosine moiety. N1 and N3 from the adenine ring form close contacts with water molecules. The O-2′ and O-3′ hydroxy groups of ribose are recognized by Asp399 through hydrogen bonds. The ribose O-4′ and O-5′ oxygen atoms are hydrogen-bonded with conserved Lys500. The sulfamoyl moiety appears to be anchored to the hydroxy group of Thr313 and a water molecule via two hydrogen bonds. The binding site of the amino acyl moiety includes two charged residues, Asp216 and Lys500, which are oriented to make contact with the amino and carboxy groups, respectively. The α-amino group of the L-alanyl moiety is involved in two salt bridge interactions (2.9 Å and 3.1 Å) with the side-chain of Asp216, and two hydrogen bonds (2.9 Å and 2.7 Å) with the backbone oxygen atoms of Gly311 and Ile317. The α-carboxy group of L-Ala-SA forms an ionic interaction with the side-chain of Lys500, which is the only residue from the C-terminal domain that directly interacts with the substrate. The Asp216 and Lys500 residues are strictly invariant in amino acid adenylation enzymes and belong to the specificity-conferring code. The other residues that are involved in substrate interaction are less conserved and are dependent on the substrate properties (e.g. polarity and size). The IdnL7 substrate binding pocket is surrounded by six nonpolar residues (Phe220, Phe256, Leu283, Ala285, Leu309 and Ile317) and two polar residues (Cys217 and Thr318). Ala285 is oriented toward the methyl group of L-Ala-SA and stabilizes the position of the L-alanyl moiety through hydrophobic interactions. Orientation of L-Ala-SA is also controlled by Phe215, which is a residue from outside the specificity-conferring code. Phe215 adjusts the Cα position of the alanyl moiety by van der Waals interactions. It should be noted that IdnL7 contains the relatively small Thr318 residue at the substrate binding pocket, providing space in front of the methyl group of alanyl moiety. This structural observation suggests that IdnL7 can also accommodate the side-chain of other small L-amino acid substrates, such as L-serine, which is consistent with the relatively relaxed substrate specificity of IdnL7. PDB reference: IdnL7, 6akd. References
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