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Mechanistic and Crystallographic Studies of Azoreductase AzoA from Bacillus wakoensis A01
- Source :
- ACS chemical biology, 15(2):acschembio.9b00970, 504-512. AMER CHEMICAL SOC, ACS Chemical Biology
- Publication Year :
- 2020
-
Abstract
- The azoreductase AzoA from the alkali-tolerant Bacillus wakoensis A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C. The dimeric enzyme contains FMN as a prosthetic group and is strictly NADH dependent. While AzoA shows a negligible ability to use molecular oxygen as an electron acceptor, it is efficient in reducing various azo dyes and quinones. The kinetic and catalytic mechanism has been studied in detail using steady state kinetic analyses and stopped-flow studies. The data show that AzoA performs quinone and azo dye reductions via a two-electron transfer. Moreover, quinones were shown to be much better substrates (kcat values of 100-400 s-1 for several naphtoquinones) when compared with azo dyes. This suggests that the physiological role of AzoA and sequence-related microbial reductases is linked to quinone reductions and that they can better be annotated as quinone reductases. The structure of AzoA has been determined in complex with FMN at 1.8 Å resolution. AzoA displays unique features in the active site providing clues for explaining its catalytic and thermostability features. An uncommon loop, when compared with sequence-related reductases, forms an active site lid with Trp60 acting as an anchor. Several Trp60 mutants have been analyzed disclosing an important role of this residue in the stability of AzoA, while they retained activity. Structural details are discussed in relation to other azo and quinone reductases. This study provides new insights into the molecular functioning of AzoA and sequence-related reductases.
- Subjects :
- 0301 basic medicine
Flavin Mononucleotide
Stereochemistry
Bacillus
Crystallography, X-Ray
01 natural sciences
Biochemistry
Catalysis
Cofactor
INDIGO
03 medical and health sciences
Quinone Reductases
Bacterial Proteins
Oxidoreductase
Catalytic Domain
NADH, NADPH Oxidoreductases
Enzyme kinetics
SPECIFICITY
Enzyme Assays
chemistry.chemical_classification
biology
010405 organic chemistry
ACTIVE-SITE
Active site
Articles
General Medicine
Nitroreductases
Electron acceptor
NAD
Recombinant Proteins
0104 chemical sciences
Quinone
PSEUDOMONAS-PUTIDA
Kinetics
030104 developmental biology
chemistry
Mutation
Mutagenesis, Site-Directed
biology.protein
Molecular Medicine
Steady state (chemistry)
Subjects
Details
- Language :
- English
- ISSN :
- 15548929
- Volume :
- 15
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- ACS chemical biology
- Accession number :
- edsair.doi.dedup.....1bd2dcf75590c727db9d62b4a2843c64