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. 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. Isonitrile, an electron-rich functional group, is a hallmark of a variety of natural products such as xanthocillin and rhabduscin and plays a role in diverse processes such as metal acquisition and virulence (Fig. S1) (1Clarke-Pearson M.F. Brady S.F. Paerucumarin, a new metabolite produced by the pvc gene cluster from Pseudomonas aeruginosa.J. Bacteriol. 2008; 190: 6927-6930Crossref PubMed Scopus (35) Google Scholar). The biosynthesis of the isonitrile group was thought to be restricted to the isonitrile synthase (IsnA) enzyme family. These enzymes catalyze the formation of isonitriles using the nitrogen atom of an α-amino group of l-Trp/l-Tyr and a carbon atom from ribulose-5-phosphate (Fig. S1) (2Brady S.F. Clardy J. Cloning and heterologous expression of isocyanide biosynthetic genes from environmental DNA.Angew. Chem. PubMed Scopus Google Scholar). of a gene cluster in some a biosynthetic pathway for isonitrile as part of the formation of isonitrile a of the gene cluster from Streptomyces that isonitrile biosynthesis is by the enzyme ScoE J. J. J. of isonitrile by nonribosomal gene in PubMed Scopus Google Scholar). the ScoE forms an isonitrile moiety from a a to a as by J. J. J. of isonitrile by nonribosomal gene in PubMed Scopus Google (Fig. ScoE a enzymatic mechanism of isonitrile biosynthesis J. J. J. of isonitrile by nonribosomal gene in PubMed Scopus Google Scholar). of the gene that the gene a α-ketoglutarate dioxygenase J. J. J. of isonitrile by nonribosomal gene in PubMed Scopus Google Scholar). This was it was that both and by ScoE in for isonitrile formation an substrate, acid (CABA), to an acid (Fig. J. formation by a Chem. PubMed Scopus Google Scholar). This ScoE in the a of enzymes that a mononuclear Fe(II) and to catalyze a diverse of such as and of and Chem. PubMed Scopus Google Scholar). isonitrile formation is a new in this enzyme family. of the are thought to a mechanism of (Fig. of and Chem. PubMed Scopus Google Scholar). This the mononuclear Fe(II) which is by a for that as to the The cosubstrate the in a one site, found in to the binding site and PubMed Scopus Google Scholar). This site is the binding site of which is with one atom into to and carbon and the atom to the to a Fe(IV)=O for the mechanism of to and of PubMed Scopus Google J. the for dioxygenase using PubMed Scopus Google The of a in the reaction of an a in dioxygenase from PubMed Scopus Google (Fig. This reaction is thought to in the of substrate, it has been in the of as a of the J. PubMed Scopus Google Scholar). The Fe(IV)=O a atom from the to an and a (Fig. for from of by the by dioxygenase Chem. PubMed Scopus Google Scholar). The of this enzymes of this family. The reaction of this enzyme is in which the is such that the group to the to a and the Fe(II) (Fig. of α-ketoglutarate dioxygenase from J. Chem. Scopus Google Scholar). The is the and is to be the enzyme of the of and Chem. PubMed Scopus Google Scholar). of the reaction by ScoE (Fig. has to the that ScoE as part of the of (Fig. J. formation by a Chem. PubMed Scopus Google J. J. from to by and Chem. PubMed Scopus Google Scholar). The of the are the of the glycyl moiety has been to be both and and the nitrogen of the glycyl moiety has been to be a site of (Fig. and a as an in the reaction from from Streptomyces a by using a J. J. from to by and Chem. PubMed Scopus Google Scholar). this was and in to it be to by of the of two of and two of and found that of produced for of isonitrile by ScoE and that this be to of from J. J. from to by and Chem. PubMed Scopus Google Scholar). Here, we the stoichiometry of this reaction and that one is and one is two of and of are of ScoE been The crystal structure of ScoE the a known as the that it is with this of enzymes J. formation by a Chem. PubMed Scopus Google Scholar). The structure the for Fe(II) binding J. formation by a Chem. PubMed Scopus Google Scholar). this structure and the with and of and J. formation by a Chem. PubMed Scopus Google Scholar). a crystal structure of ScoE to and the Å J. J. from to by and Chem. PubMed Scopus Google Scholar). Here, we present a structure of ScoE with Fe(II) and Å and binding with the protein to crystal structures of ScoE with in an that the binding structural the ScoE structures to a that the residues and by approximately 10 Å in the active site, to be an binding site for that the binding with a crystal structure of ScoE with a mimic of the we how structural, biochemical, and computational data of this unprecedented reaction for this enzyme superfamily. 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