Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase

2014 ◽  
Vol 462 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Akito Nishizawa ◽  
Ayaka Harada ◽  
Miki Senda ◽  
Yuka Tachihara ◽  
Daisuke Muramatsu ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Pyridine Nucleotide ◽  
Biphenyl Dioxygenase ◽  
Ferredoxin Reductase ◽  
Dependent Form

An NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis.

Crystal structure of NADH-dependent ferredoxin reductase component in biphenyl dioxygenase

2000 ◽  
Vol 304 (3) ◽  
pp. 397-410 ◽  
Author(s):  
Toshiya Senda ◽  
Takahiro Yamada ◽  
Nobuyuki Sakurai ◽  
Miho Kubota ◽  
Tomoko Nishizaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biphenyl Dioxygenase ◽  
Ferredoxin Reductase

scholarly journals Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA

2003 ◽  
Vol 372 (2) ◽  
pp. 317-327 ◽  
Author(s):  
Kirsty J. McLEAN ◽  
Nigel S. SCRUTTON ◽  
Andrew W. MUNRO
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
Spectral Characteristics ◽  
Reduction Rate ◽  
Pyridine Nucleotide ◽  
Cytochrome P450 Enzymes ◽  
Hydrogen Electrode ◽  
Ferricyanide Reduction ◽  
Ferredoxin Reductase ◽  
Adrenodoxin Reductase

The genome sequence of the pathogenic bacterium Mycobacterium tuberculosis revealed numerous cytochrome P450 enzymes, which require accessory redox enzymes for catalytic function (ferredoxin reductase and ferredoxin). The most likely ferredoxin reductase is encoded by fprA, and its structure resembles eukaryotic adrenodoxin reductases. We have cloned, expressed and purified the flavoenzyme product of the fprA gene in Escherichia coli. FprA reduces various electron acceptors using either NADPH or NADH as the electron donor, but discriminates in favour of NADPH (apparent Km for NADH=50.6±3.1 μM; NADPH=4.1±0.3 μM from ferricyanide reduction experiments). Stopped-flow studies of reduction of the FprA FAD by NADPH demonstrate increased flavin reduction rate at low NADPH concentration (<200 μM), consistent with the presence of a second, kinetically distinct and inhibitory, pyridine nucleotide-binding site, similar to that identified in human cytochrome P450 reductase [Gutierrez, Lian, Wolf, Scrutton and Roberts (2001) Biochemistry 40, 1964–1975]. Flavin reduction by NADH is slower than with NADPH and displays hyperbolic dependence on NADH concentration [maximal reduction rate (kred)=25.4±0.7 s−1, apparent Kd=42.9±4.6 μM]. Flavin reoxidation by molecular oxygen is more rapid for NADH-reduced enzyme. Reductive titrations show that the enzyme forms a species with spectral characteristics typical of a neutral (blue) FAD semiquinone only on reduction with NADPH, consistent with EPR studies. The second order dependence of semiquinone formation on the concentration of FprA indicates a disproportionation reaction involving oxidized and two-electron-reduced FprA. Titration of FprA with dithionite converts oxidized FAD into the hydroquinone form; the flavin semiquinone is not populated under these conditions. The midpoint reduction potential for the two electron couple is −235±5 mV (versus the normal hydrogen electrode), similar to that for adrenodoxin reductase (−274 mV). Our data provide a thermodynamic and transient kinetic framework for catalysis by FprA, and complement recent spectrophotometric and steady-state studies of the enzyme [Fischer, Raimondi, Aliverti and Zanetti (2002) Eur. J. Biochem. 269, 3005–3013].

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

Studying the function of methyltransferases using nucleotide analogue based random mutagenesis

1999 ◽  
Author(s):  
Victoria H. Harris ◽  
Alan Hamilton ◽  
David M. Williams ◽  
David P. Hornby
Keyword(s):  
Random Mutagenesis ◽  
Nucleotide Analogue

scholarly journals Development of Engineered Ferredoxin Reductase Systems for the Efficient Hydroxylation of Steroidal Substrates

2020 ◽  
Vol 8 (44) ◽  
pp. 16720-16730
Author(s):  
Zhangliang Zhu ◽  
Xin Gao ◽  
Zhan Song ◽  
Chao Li ◽  
Fuping Lu ◽  
...  
Keyword(s):  
Ferredoxin Reductase
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