Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase

Abstract

The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45-1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an "inducible" α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.

Keywords: crystallography; enzyme mechanism; molecular dynamics; mononuclear Fe(II) α-ketoglutarate dependent dioxygenase; stoichiometry; structure function.

Figure 1

Reaction catalyzed by ScoE.A, ScoE is part of a nonribosomal peptide biosynthetic pathway that makes an isonitrile lipopeptide (INLP) and acts on an untethered substrate. B, isotope labeling studies have confirmed that the carbon–nitrogen single bond in CABA is converted to the isonitrile moiety. The full four-electron oxidation is catalyzed by the Fe(II)/αKG dioxygenase ScoE. The carbon numbering is indicated.

Figure 2

Possible mechanisms of isonitrile formation by ScoE. Top schemes (i and ii) show αKG-dependent hydroxylation at C5 of CABA occurring first. Schemes i and ii differ in the second half-reaction with αKG. These schemes are based on reaction mechanisms proposed by Chang and coworkers (13). In scheme iii, αKG-dependent hydroxylation of N occurs before αKG-dependent hydroxylation of C5 of CABA.

Figure 3

Active site comparisons.A, ScoE with CABA (cyan), Fe(II) (orange sphere), and acetate (purple). For clarity, the nitrogen atom of CABA is labeled. B, TauD (1OS7) with taurine (red), α-KG (purple), Fe(II) (orange sphere), and facial triad (wheat). C, ScoE with CABA (cyan) and oxovanadium (gray with oxygen atom in red). D, superposition of ScoE structures shows two positions of Arg157 and His299. The structures solved in this work are shown in yellow, the previously determined structure (with tartrate bound, PDB: 6L6X) is shown in green. CABA is shown in cyan, tartrate in purple, and Fe(II) as an orange sphere. Maps shown in blue mesh above are 2F_o−F_c composite omit maps contoured at 1 σ. Dashed lines indicate close interactions (less than 4.0 Å).

Figure 4

Comparison of ScoE surface at Lys193 in the presence of choline and CABA.A, surface of ScoE protein with choline, chloride, and Zn(II) in the active site (PDB: 6DCH). Choline is shown in green, chloride is shown as a green sphere, and Zn(II) is shown as a purple sphere. The protein surface is represented in gray. Lys193 is labeled and is flipped out, exposing the active site to solvent. B, surface of ScoE protein with CABA and Fe(II) bound in the active site. CABA is shown in cyan and Fe(II) is shown as an orange sphere. The protein surface is represented in gray. Lys193 is flipped in to make a hydrogen bond with CABA and close the active site.

Figure 5

Hydrogen bonding network in ScoE extends from CABA to the protein surface.A, CABA is shown in cyan, acetate is shown in purple, and Fe(II) is shown as an orange sphere. Tyr96, Tyr101, and Arg195 are shown in gray, indicating that product is not detected when these residues are substituted. Tyr97 is shown in red to indicate that substitution of this residue results in reduced product formation. Dashed lines indicate close interactions (less than 4.0 Å). For clarity, the nitrogen atom of CABA is labeled. B, extracted ion chromatograms corresponding to the production of the INLP (calculated:[M + H]+= 323.2078, observed:[M + H]+= 323.2093, 4.6 ppm error) from coupled ScoABCE assays using wild-type ScoE and four single amino acid variants. In all cases, the calculated masses with a 10-ppm error tolerance were used. A reaction mixture comprised of 50 mM HEPES, pH 8, 500 μM α-KG, 2 mM CABA, 100 μM Apo-ScoE, and 90 μM (NH₄)₂Fe(SO₄)₂ was gently mixed and incubated for 10 min at room temperature. After incubation, the reaction was mixed with 25 μl of 2 mM MgCl₂, 5 mM ATP, 500 μM lysine, 4 mM NADPH, 50 μM ScoA, 50 μM ScoB, and 20 μM ScoC.

Figure 6

Movement of Arg157 and His299 in the absence and presence of tartrate in the ScoE active site appears to be relevant for α-ketoglutarate binding.A, active site view of ScoE with CABA (cyan), Fe(II) (orange sphere), and acetate (purple) showing loop regions containing His299 and Arg 157, both flipped away from the active site. This loop conformation is stabilized by side chain to backbone hydrogen bonds (black dashes). B, surface of ScoE structure shown in panel A. When His299 and Arg157 are positioned away from the active site, the active site is open and accessible for αKG binding. C, active site view of ScoE (PDB: 6L6X) with Fe(II) (orange sphere) and tartrate (green), showing loop regions containing His299 and Arg157. His299 and Arg157 are positioned toward the active site and form a stacking interaction. This conformation is also stabilized by backbone interactions between the loops (black dashes). D, surface of ScoE structure shown in panel C (PDB: 6L6X). When His299 and Arg157 are flipped in, the active site is closed and no longer solvent accessible. E, extracted ion chromatograms corresponding to the production of Py-aminopyrazole (calculated:[M + H]+= 238.1087, observed:[M + H]+= 238.1095, 3.4 ppm error) from ScoE assays using wild-type ScoE and three single amino acid variants. In all cases, the calculated masses with a 10-ppm error tolerance were used. A reaction mixture comprised of 50 mM HEPES, pH 8, 500 μM CABA, 250 μM α-KG, 100 μM Apo-ScoE, and 90 μM (NH₄)2Fe(SO₄)₂ was gently mixed and incubated for 10 min at room temperature, which was further quenched with 200 μl of 667 μM 3,6-di(pyridine-2-yl)-1,2,4,5-tetrazine dissolved in cold methanol. 3,6-Di-2-pyridyl-1,2,4,5-tetrazine reacts with ScoE product INBA to generate Py-aminopyrazole. F, extracted ion chromatograms corresponding to the production of succinate (calculated:[M + H]+= 117.0193, observed:[M + H]+= 117.0193, 0 ppm error) from ScoE assays using wild-type ScoE and three single amino acid variants. In all cases, the calculated masses with a 10-ppm error tolerance were used. The same reaction mixture as used in E was used for this analysis. G, extracted ion chromatograms corresponding to the production of succinate (calculated:[M + H]+= 117.0193, observed:[M + H]+= 117.0193, 0 ppm error) from ScoE assays lacking CABA using wild-type ScoE and three single amino acid variants. In all cases, the calculated masses with a 10-ppm error tolerance were used. A reaction mixture comprised of 50 mM HEPES, pH 8, 250 μM α-KG, 100 μM Apo-ScoE, and 90 μM (NH₄)₂Fe(SO₄)₂ was gently mixed and incubated for 10 min at room temperature, which was further quenched with 200 μl of 667 μM 3,6-di(pyridine-2-yl)-1,2,4,5-tetrazine dissolved in cold methanol.

Figure 7

Proposed inducible motions in ScoE that form binding sites for the cosubstrates during the ScoE reaction. CABA binding is stabilized by a conformational change to bring the side chain of Lys193 into the active site to interact with one end of the CABA molecule. The side chains of His299 and Arg157 must swing a distance of 10 Å toward the active site to form the α-KG binding site and swing out again to release succinate and CO₂. These side chains must swing back into the active site to form the binding site for the second molecule of α-KG. Upon formation of the INBA product, Lys193, His299, and Arg157 must all flip out of the active site to facilitate product release. Note: although CABA is shown as hydroxylated on C5, the site of the first (or second) hydroxylation is not firmly established.