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Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa - decarboxylation is not essential for indole formation

DOI: 10.1074/jbc.RA120.014936 DOI Help
PMID: 32928960 PMID Help

Authors: Annika Soderholm (Uppsala University) , Matilda S. Newton (University of Otago) , Wayne M. Patrick (Victoria University of Wellington) , Maria Selmer (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 295(47) , PAGES 15948-15956

State: Published (Approved)
Published: November 2020
Diamond Proposal Number(s): 15868 , 22906

Open Access Open Access

Abstract: In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGP synthase, IGPS) starts with a condensation step where the substrate’s carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation which restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. This step was since the 1960s considered to be mechanistically essential based on studies of the IGPS:PRAI fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a non-reactive substrate analogue, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate (PAdRP) – with approximately 1000' lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, out of which seven contain ligand, and one displays a previously unobserved conformation closer to the reactive state. One of the few non-conserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that, despite IGPS’s classification as a carboxylyase (i.e. decarboxylase), decarboxylation not a completely essential step in its catalysis.

Journal Keywords: enzyme promiscuity; IGPS; tryptophan biosynthesis; decarboxylase; enzyme kinetics; enzyme catalysis; enzyme structure; enzyme mechanism; biosynthesis

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: B21-High Throughput SAXS , I04-Macromolecular Crystallography

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