Journal:Acta Cryst F:S2053230X19002863
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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. | 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. | ||
| - | <scene name='80/809196/Cv/2'>Cartoon representation of the overall structure of IdnL7</scene>. The overall structure of IdnL7 consists of two domains: a large <scene name='80/809196/Cv/30'>N-terminal domain</scene> (Met1–Gly413; in pink) and a smaller <scene name='80/809196/Cv/31'>C-terminal domain</scene> (Gln420–Leu522; in magenta). Both domains are connected by a <scene name='80/809196/Cv/29'>flexible hinge region</scene> (Arg414–Leu419), which allows for the rotation of the C-terminal domain during the two catalytic reaction steps. The N-terminal domain forms a <scene name='80/809196/Cv/33'>five-layered αβαβα-sandwich fold</scene>, whereas the C-terminal domain comprises <scene name='80/809196/Cv/34'>three helices with one two-stranded and one three-stranded antiparallel β-sheet</scene>. The C-terminal domain is arranged in the adenylation conformation in relation to the N-terminal domain. | + | <scene name='80/809196/Cv/2'>Cartoon representation of the overall structure of IdnL7</scene> (PDB entry [[6akd]]). The overall structure of IdnL7 consists of two domains: a large <scene name='80/809196/Cv/30'>N-terminal domain</scene> (Met1–Gly413; in pink) and a smaller <scene name='80/809196/Cv/31'>C-terminal domain</scene> (Gln420–Leu522; in magenta). Both domains are connected by a <scene name='80/809196/Cv/29'>flexible hinge region</scene> (Arg414–Leu419), which allows for the rotation of the C-terminal domain during the two catalytic reaction steps. The N-terminal domain forms a <scene name='80/809196/Cv/33'>five-layered αβαβα-sandwich fold</scene>, whereas the C-terminal domain comprises <scene name='80/809196/Cv/34'>three helices with one two-stranded and one three-stranded antiparallel β-sheet</scene>. The C-terminal domain is arranged in the adenylation conformation in relation to the N-terminal domain. |
<scene name='80/809196/Cv/11'>Ligand binding site</scene>. 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 <scene name='80/809196/Cv/38'>Tyr310, Tyr411 and Ile340 on one side, and a loop (residues Gly286–Ala288)</scene> on the other side. This architecture stabilizes the purine base by hydrophobic and van der Waals interactions. Additionally, the <scene name='80/809196/Cv/39'>N6 amino group of the adenine ring interacts with the main chain carbonyl of Leu309 and the side-chain amide group of Asn308</scene>. Several water molecules participate in direct interactions with the sulfamoyladenosine moiety. <scene name='80/809196/Cv/40'>N1 and N3 from the adenine ring form close contacts with water molecules</scene>. The <scene name='80/809196/Cv/42'>O-2′ and O-3′ hydroxy groups of ribose are recognized by Asp399</scene> through hydrogen bonds. The ribose <scene name='80/809196/Cv/43'>O-4′ and O-5′ oxygen atoms are hydrogen-bonded with conserved Lys500</scene>. The <scene name='80/809196/Cv/44'>sulfamoyl moiety appears to be anchored to the hydroxy group of Thr313 and a water molecule via two hydrogen bonds</scene>. | <scene name='80/809196/Cv/11'>Ligand binding site</scene>. 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 <scene name='80/809196/Cv/38'>Tyr310, Tyr411 and Ile340 on one side, and a loop (residues Gly286–Ala288)</scene> on the other side. This architecture stabilizes the purine base by hydrophobic and van der Waals interactions. Additionally, the <scene name='80/809196/Cv/39'>N6 amino group of the adenine ring interacts with the main chain carbonyl of Leu309 and the side-chain amide group of Asn308</scene>. Several water molecules participate in direct interactions with the sulfamoyladenosine moiety. <scene name='80/809196/Cv/40'>N1 and N3 from the adenine ring form close contacts with water molecules</scene>. The <scene name='80/809196/Cv/42'>O-2′ and O-3′ hydroxy groups of ribose are recognized by Asp399</scene> through hydrogen bonds. The ribose <scene name='80/809196/Cv/43'>O-4′ and O-5′ oxygen atoms are hydrogen-bonded with conserved Lys500</scene>. The <scene name='80/809196/Cv/44'>sulfamoyl moiety appears to be anchored to the hydroxy group of Thr313 and a water molecule via two hydrogen bonds</scene>. | ||
The binding site of the amino acyl moiety includes two charged residues, <scene name='80/809196/Cv/45'>Asp216 and Lys500</scene>, 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 <scene name='80/809196/Cv/46'>Asp216</scene>, and two hydrogen bonds (2.9 Å and 2.7 Å) with the backbone oxygen atoms of <scene name='80/809196/Cv/47'>Gly311 and Ile317</scene>. The α-carboxy group of L-Ala-SA forms an ionic interaction with the side-chain of <scene name='80/809196/Cv/48'>Lys500</scene>, 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 (<scene name='80/809196/Cv/49'>Phe220, Phe256, Leu283, Ala285, Leu309 and Ile317</scene>) and two polar residues (<scene name='80/809196/Cv/50'>Cys217 and Thr318</scene>). <scene name='80/809196/Cv/53'>Ala285</scene> 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 <scene name='80/809196/Cv/51'>Phe215</scene>, 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 <scene name='80/809196/Cv/52'>Thr318</scene> 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. | The binding site of the amino acyl moiety includes two charged residues, <scene name='80/809196/Cv/45'>Asp216 and Lys500</scene>, 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 <scene name='80/809196/Cv/46'>Asp216</scene>, and two hydrogen bonds (2.9 Å and 2.7 Å) with the backbone oxygen atoms of <scene name='80/809196/Cv/47'>Gly311 and Ile317</scene>. The α-carboxy group of L-Ala-SA forms an ionic interaction with the side-chain of <scene name='80/809196/Cv/48'>Lys500</scene>, 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 (<scene name='80/809196/Cv/49'>Phe220, Phe256, Leu283, Ala285, Leu309 and Ile317</scene>) and two polar residues (<scene name='80/809196/Cv/50'>Cys217 and Thr318</scene>). <scene name='80/809196/Cv/53'>Ala285</scene> 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 <scene name='80/809196/Cv/51'>Phe215</scene>, 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 <scene name='80/809196/Cv/52'>Thr318</scene> 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. | ||
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| + | '''PDB reference:''' IdnL7, [[6akd]]. | ||
<b>References</b><br> | <b>References</b><br> | ||
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