redox partner
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2021 ◽  
Vol 22 (24) ◽  
pp. 13451
Author(s):  
Anna M. Banaś ◽  
Katarzyna M. Bocian-Ostrzycka ◽  
Stanisław Dunin-Horkawicz ◽  
Jan Ludwiczak ◽  
Piotr Wilk ◽  
...  

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.


AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thomas Hilberath ◽  
Alessandra Raffaele ◽  
Leonie M. Windeln ◽  
Vlada B. Urlacher

AbstractCytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.


2021 ◽  
Author(s):  
Xiaohui Liu ◽  
Fengwei Li ◽  
Tianjian Sun ◽  
Jiawei Guo ◽  
Xingwang Zhang ◽  
...  

Abstract Cytochrome P450 enzymes (P450s) are highly attractive biocatalysts due to their versatile catalytic activities. A vast majority of P450s require redox partner (RP) proteins to sequentially transfer two electrons for O2 activation and substrate oxidation. However, little information is available on cognate RPs for P450s, which greatly limits P450 function exploration and practical application. Thus, the stategy of building various hybrid P450 catalytic systems with surrogate RPs has often adopted to engineer P450 biocatalysts for different purposes. In this study, we comprehensively compare three pairs of frequently-used surrogate redox partners SelFdx1499/SelFdR0978, Adx/AdR and Pdx/PdR and in terms of their electron transfer properties. The three selected bacterial Class I P450s to accept electrons from RPs include PikC, P450sca-2 and CYP-sb21, which are responsible for production of macrolide antibiotics, the cholesterol-lowering drug pravastatin, and a hair-growth-stimulating agent. Both experimental studies and structural analysis show that SelFdx1499/SelFdR0978 is the most promising RP compared to Adx/AdR and Pdx/PdR. The results provide insights into the domination for P450-redox partner interactions in modulating the catalytic activity of P450s. This study not only produces a more active biocatalyst but also suggests a general chose for a universal reductase which would facilitate engineering of P450 catalyst.


2021 ◽  
Author(s):  
Sarah Wolfson ◽  
Reese Hitchings ◽  
Karina Peregrina ◽  
Ziv Cohen ◽  
Saad Khan ◽  
...  

Abstract Although microbial biochemistry shapes a dynamic environment in the gut, how bacterial metabolites such as hydrogen sulfide (H2S) mechanistically alter the gut chemical landscape is poorly understood. Here we show for the first time that H2S generated during cysteine metabolism drives the reduction of azo (R-N=N-R’) xenobiotics in bacterial cultures, human fecal microbial communities, and in vivo mouse models. Thus, chemical-chemical interactions, derived from microbial community metabolism, are a key missing feature shaping xenobiotic metabolism in the gut. Changing dietary levels of the H2S xenobiotic redox partner Red 40 transiently decreases mouse fecal sulfide, confirming that a xenobiotic can attenuate sulfide concentration in vivo. Cryptic H2S redox thus modulates sulfur homeostasis in the gut and the fate of xenobiotics to which humans are regularly exposed.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1429
Author(s):  
Rowoon Lee ◽  
Vitchan Kim ◽  
Youngjin Chun ◽  
Donghak Kim

The human genome includes four cytochrome P450 2C subfamily enzymes, and CYP2C8 has generated research interest because it is subject to drug–drug interactions and various polymorphic outcomes. To address the structure-functional complexity of CYP2C8, its catalytic activity was studied using a directed evolution analysis. Consecutive rounds of random mutagenesis and screening using 6-methoxy-luciferin produced two mutants, which displayed highly increased luciferase activity. Wild-type and selected mutants were expressed on a large scale and purified. The expression levels of the D349Y and D349Y/V237A mutants were ~310 and 460 nmol per liter of culture, respectively. The steady-state kinetic analysis of paclitaxel 6α-hydroxylation showed that the mutants exhibited a 5–7-fold increase in kcat values and a 3–5-fold increase in catalytic efficiencies (kcat/KM). In arachidonic acid epoxidation, two mutants exhibited a 30–150-fold increase in kcat values and a 40–110-fold increase in catalytic efficiencies. The binding titration analyses of paclitaxel and arachidonic acid showed that the V237A mutation had a lower Kd value, indicating a tighter substrate-binding affinity. The structural analysis of CYP2C8 indicated that the D349Y mutation was close enough to the putative binding domain of the redox partner; the increase in catalytic activity could be partially attributed to the enhancement of the P450 coupling efficiency or electron transfer.


2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ashkan Habib ◽  
Alireza Shojazadeh ◽  
Mohadeseh Molayemat ◽  
Hossein Jafari Khamirani ◽  
Sina Zoghi ◽  
...  

AbstractIn this study, we detected homozygous mutations in the CYP17A1 gene (NM_000102.4:c.1053_1055delCCT; p.Leu353del; SCV001479329) in a 28-year-old female patient (46,XX) and her phenotypically female 30-year-old sister (46,XY) who had phenotypes consistent with combined 17-hydroxylase and 17,20-lyase deficiency. The phenotypes were not expected based on the location of the mutation in the CYP17A1 redox partner-binding site and a previous description of the same mutation linked with isolated 17,20-lyase deficiency.


Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 913
Author(s):  
Jorge Guío ◽  
María Teresa Bes ◽  
Mónica Balsera ◽  
Laura Calvo-Begueria ◽  
Emma Sevilla ◽  
...  

FurA is a multifunctional regulator in cyanobacteria that contains five cysteines, four of them arranged into two CXXC motifs. Lack of a structural zinc ion enables FurA to develop disulfide reductase activity. In vivo, FurA displays several redox isoforms, and the oxidation state of its cysteines determines its activity as regulator and its ability to bind different metabolites. Because of the relationship between FurA and the control of genes involved in oxidative stress defense and photosynthetic metabolism, we sought to investigate the role of type m thioredoxin TrxA as a potential redox partner mediating dithiol-disulfide exchange reactions necessary to facilitate the interaction of FurA with its different ligands. Both in vitro cross-linking assays and in vivo two-hybrid studies confirmed the interaction between FurA and TrxA. Light to dark transitions resulted in reversible oxidation of a fraction of the regulator present in Anabaena sp. PCC7120. Reconstitution of an electron transport chain using E. coli NADPH-thioredoxin-reductase followed by alkylation of FurA reduced cysteines evidenced the ability of TrxA to reduce FurA. Furthermore, the use of site-directed mutants allowed us to propose a plausible mechanism for FurA reduction. These results point to TrxA as one of the redox partners that modulates FurA performance.


2021 ◽  
Author(s):  
Bastien Bissaro ◽  
Sayo Kodama ◽  
Hayat Hage ◽  
David Ribeaucourt ◽  
Mireille Haon ◽  
...  

Abstract Copper radical oxidases (CRO) form a class of enzymes with a longstanding history encompassing diverse substrate specificities. While the biological function of most CROs remains unknown, we observed that CROs active on aliphatic alcohols are found only in fungal plant pathogens. Here, we unveil the role of these CROs and the identity of their natural redox partner, a haem-iron peroxidase. Combining multiscale approaches, we report that Colletotrichum and Magnaporthe appressoria (specialized cells that puncture the plant cuticles) co-secrete this pair of metalloenzymes early during penetration. We show in vivo that mutant appressoria lacking either or both enzymes have impaired penetration ability and pathogenicity. We reveal in vitro a finely-tuned enzyme interplay is responsible for the oxidation of plant cuticular long-chain alcohols into aldehyde products, suggested to act as key molecular signals in the fungal infection machinery. Our results open new avenues to design oxidase-specific inhibitors as anti-penetrants for crop protection.


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