5G3S

The structure of the L-tryptophan oxidase VioA from Chromobacterium violaceum - Samarium derivative

Summary for 5G3S

• Entry DOI: 10.2210/pdb5g3s/pdb
• Related: 5G3T 5G3U
• Descriptor: L-TRYPTOPHAN OXIDASE VIOA, SAMARIUM (III) ION, MERCURY (II) ION, ... (8 entities in total)
• Functional Keywords: oxidoreductase, violacein, l-tryptophan oxidase, flavoenzyme, samarium derivative
• Biological source: CHROMOBACTERIUM VIOLACEUM
• Total number of polymer chains: 2
• Total formula weight: 100788.19

Authors

Krausze, J.,Rabe, J.,Moser, J. (deposition date: 2016-05-01, release date: 2016-08-03, Last modification date: 2024-11-20)

Primary citation

Fuller, J.,Roepke, R.,Krausze, J.,Rennhack, K.E.,Daniel, N.P.,Blankenfeldt, W.,Schulz, S.,Jahn, D.,Moser, J.
Biosynthesis of Violacein: Structure and Function of L-Tryptophan Oxidase Vioa Chromobacterium Violaceum
J.Biol.Chem., 291:20068-, 2016

PubMed Abstract: Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg(64), Lys(269), and Tyr(309) as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His(163) in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.

PubMed: 27466367
DOI: 10.1074/JBC.M116.741561

Experimental method

X-RAY DIFFRACTION (2.076 Å)