Journal:Acta Cryst F:S2053230X19002863

Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis

Jolanta Cieślak, Akimasa Miyanaga, Makoto Takaishi, Fumitaka Kudo, Tadashi Eguchi ^[1]

Molecular Tour
Adenylation enzymes are responsible for the selective incorporation of an appropriate carboxylate building block in the biosynthesis of nonribosomal peptides and related natural products. The substrate specificity of adenylation enzymes dictates the chemical structure of natural products. Understanding how adenylation enzymes recognize their own substrates is important for engineering biosynthetic pathways to produce novel compounds for drug discovery.

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.

. The overall structure of IdnL7 consists of two domains: a large (Met1–Gly413; in pink) and a smaller (Gln420–Leu522; in magenta). Both domains are connected by a (Arg414–Leu419), which allows for the rotation of the C-terminal domain during the two catalytic reaction steps. The N-terminal domain forms a , whereas the C-terminal domain comprises . The C-terminal domain is arranged in the adenylation conformation in relation to the N-terminal domain.

. 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 on the other side. This architecture stabilizes the purine base by hydrophobic and van der Waals interactions. Additionally, the . Several water molecules participate in direct interactions with the sulfamoyladenosine moiety. . The through hydrogen bonds. The ribose . The .

The binding site of the amino acyl moiety includes two charged residues, , 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 , and two hydrogen bonds (2.9 Å and 2.7 Å) with the backbone oxygen atoms of .

References

1. ↑ Cieslak J, Miyanaga A, Takaishi M, Kudo F, Eguchi T. Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis. Acta Crystallogr F Struct Biol Commun. 2019 Apr 1;75(Pt 4):299-306. doi:, 10.1107/S2053230X19002863. Epub 2019 Apr 2. PMID:30950831 doi:http://dx.doi.org/10.1107/S2053230X19002863