title: Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.
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.
authors.affiliation: From the Institute of Microbiology and.
authors.forename: Janis J
authors.initials: JJ
authors.lastname: Füller
authors.affiliation: the Institute of Organic Chemistry, Technische Universität Braunschweig, Hagenring 30, D-38106 Braunschweig, and.
authors.forename: René
authors.initials: R
authors.lastname: Röpke
authors.affiliation: the Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.
authors.forename: Joern
authors.initials: J
authors.lastname: Krausze
authors.affiliation: From the Institute of Microbiology and.
authors.forename: Kim E
authors.initials: KE
authors.lastname: Rennhack
authors.affiliation: From the Institute of Microbiology and.
authors.forename: Nils P
authors.initials: NP
authors.lastname: Daniel
authors.affiliation: the Structure and Function of Proteins, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany Institute of Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig.
authors.forename: Wulf
authors.initials: W
authors.lastname: Blankenfeldt
authors.affiliation: the Institute of Organic Chemistry, Technische Universität Braunschweig, Hagenring 30, D-38106 Braunschweig, and.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulz
authors.affiliation: From the Institute of Microbiology and d.jahn@tu-bs.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
authors.affiliation: From the Institute of Microbiology and.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moser
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002861:Chromobacterium / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D000072417:Protein Domains; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007211:Indoles; D005182:Flavin-Adenine Dinucleotide; C058805:1,5-dihydro-FAD; D014364:Tryptophan; D014366:Tryptophan Oxygenase; C063155:violacein
keywords: L-tryptophan oxidase; crystal structure; enzyme mechanism; enzyme-inhibitor complex; flavoprotein; redox chemistry; site-directed mutagenesis; substrate specificity; violacein