Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.

2.50
Hdl Handle:
http://hdl.handle.net/10033/621319
Title:
Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.
Authors:
Füller, Janis J; Röpke, René; Krausze, Joern; Rennhack, Kim E; Daniel, Nils P; Blankenfeldt, Wulf ( 0000-0001-9886-9668 ) ; Schulz, Stefan; Jahn, Dieter; Moser, Jürgen
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.
Affiliation:
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum. 2016, 291 (38):20068-84 J. Biol. Chem.
Journal:
The Journal of biological chemistry
Issue Date:
2016
URI:
http://hdl.handle.net/10033/621319
DOI:
10.1074/jbc.M116.741561
PubMed ID:
27466367
Type:
Article
Language:
en
ISSN:
1083-351X
Appears in Collections:
Publications of the Dept. Structure and Functions of Proteins(SFPR)

Full metadata record

DC FieldValue Language
dc.contributor.authorFüller, Janis Jen
dc.contributor.authorRöpke, Renéen
dc.contributor.authorKrausze, Joernen
dc.contributor.authorRennhack, Kim Een
dc.contributor.authorDaniel, Nils Pen
dc.contributor.authorBlankenfeldt, Wulfen
dc.contributor.authorSchulz, Stefanen
dc.contributor.authorJahn, Dieteren
dc.contributor.authorMoser, Jürgenen
dc.date.accessioned2018-03-12T13:07:45Z-
dc.date.available2018-03-12T13:07:45Z-
dc.date.issued2016-
dc.identifier.citationBiosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum. 2016, 291 (38):20068-84 J. Biol. Chem.en
dc.identifier.issn1083-351X-
dc.identifier.pmid27466367-
dc.identifier.doi10.1074/jbc.M116.741561-
dc.identifier.urihttp://hdl.handle.net/10033/621319-
dc.description.abstractViolacein 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.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.subject.meshBacterial Proteinsen
dc.subject.meshChromobacteriumen
dc.subject.meshFlavin-Adenine Dinucleotideen
dc.subject.meshIndolesen
dc.subject.meshKineticsen
dc.subject.meshProtein Domainsen
dc.subject.meshStructure-Activity Relationshipen
dc.subject.meshTryptophanen
dc.subject.meshTryptophan Oxygenaseen
dc.titleBiosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum.en
dc.typeArticleen
dc.contributor.departmentHelmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalThe Journal of biological chemistryen

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