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Cytochrome b5 (CB5), a small heme-binding protein, plays an important role in plant biotic and abiotic stress. Anthocyanin is a critical determinant for fruit coloration, however, whether CB5 is involved in regulating anthocyanin biosynthesis has not yet been investigated in apple fruit (Malus domestica). In this study, we determined that MdCYB5, an apple CB5 gene, was a positive regulator for anthocyanin biosynthesis in apple fruit. We first found that MdCYB5 showed a high sequence and structural similarity with Arabidopsis cytochrome b5 isoform E (CB5E) at the protein level. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that MdCYB5 responds to light signals. Subcellular localization showed that MdCYB5 is localized to the cytoplasmin inthe epidermal cells of Nicotiana benthamiana leaves. Further investigation revealed that overexpressing MdCYB5 promoted anthocyanin biosynthesis in both apple calli and tissue-cultured apple seedlings. Furthermore, results of transient expression assay showed that overexpressing MdCYB5 promoted anthocyanin accumulation and fruit coloration in apple fruit. Taken together, this study suggests that MdCYB5 has a positive regulatory effect on anthocyanin biosynthesis in apple fruit. [ABSTRACT FROM AUTHOR]

SUMMARY: Flavan‐3‐ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan‐3‐ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan‐3‐ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI‐MSI method to reveal a distinct distribution pattern of B‐ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3′H, and CsF3′5′H localizes endoplasmic reticulum. Protein–protein interaction studies demonstrated direct associations between CsC4H, CsF3′H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3′5′H, the enzyme for B‐ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3′5′H. Our findings suggest the existence of distinct modules governing the synthesis of different B‐ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B‐ring trihydroxylated flavan‐3‐ols in tea plants. Significance Statement: Our work reveals the mechanism of flavonoid diversity and efficient synthesis in tea plants from the perspective of metabolon. Different metabolons modules provide a new perspective on the scientific problem of why tea plants accumulate high levels of EGCG. The discovery of obligate electron shuttle proteins provides new directions for improving metabolic engineering. [ABSTRACT FROM AUTHOR]

NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD‐Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter‐domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full‐length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high‐resolution atomic structures of the b5 domain and a construct containing the CS and b5R domains (CS/b5R). Ncb5or also contains an N‐terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N‐terminal region naturally fused to the b5 domain, we were able to obtain a high‐resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11‐residue α‐helix (S116QMDWLKLTRT126) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N‐terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi‐domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes. [ABSTRACT FROM AUTHOR]

Cytochrome b5 (CB5), a small heme-binding protein, plays an important role in plant biotic and abiotic stress. Anthocyanin is a critical determinant for fruit coloration, however, whether CB5 is involved in regulating anthocyanin biosynthesis has not yet been investigated in apple fruit (Malus domestica). In this study, we determined that MdCYB5, an apple CB5 gene, was a positive regulator for anthocyanin biosynthesis in apple fruit. We first found that MdCYB5 showed a high sequence and structural similarity with Arabidopsis cytochrome b5 isoform E (CB5E) at the protein level. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that MdCYB5 responds to light signals. Subcellular localization showed that MdCYB5 is localized to the cytoplasmin inthe epidermal cells of Nicotiana benthamiana leaves. Further investigation revealed that overexpressing MdCYB5 promoted anthocyanin biosynthesis in both apple calli and tissue-cultured apple seedlings. Furthermore, results of transient expression assay showed that overexpressing MdCYB5 promoted anthocyanin accumulation and fruit coloration in apple fruit. Taken together, this study suggests that MdCYB5 has a positive regulatory effect on anthocyanin biosynthesis in apple fruit.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein–protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes. [ABSTRACT FROM AUTHOR]

For a series of cytochrome b5 proteins isolated from various species, including bacteria, animals, and humans, we analyzed the intrinsic strength of their distal/proximal FeN bonds and the intrinsic stiffness of their axial NFeN bond angles. To assess intrinsic bond strength and bond angle stiffness, we employed local vibrational stretching force constants k a (FeN) and bending force constants k a (NFeN) derived from the local mode theory developed by our group; the ferric and ferrous oxidation states of the heme Fe were considered. All calculations were conducted with the QM/MM methodology. We found that the reduction of the heme Fe from the ferric to the ferrous state makes the FeN axial bonds weaker, longer, less covalent, and less polar. Additionally, the axial NFeN bond angle becomes stiffer and less flexible. Local mode force constants turned out to be far more sensitive to the protein environment than geometries; evaluating force constant trends across diverse protein groups and monitoring changes in the axial heme-framework revealed redox-induced changes to the primary coordination sphere of the protein. These results indicate that local mode force constants can serve as useful feature data for training machine learning models that predict cytochrome b5 redox potentials, which currently rely more on geometric data and qualitative descriptors of the protein environment. The insights gained through our investigation also offer valuable guidance for strategically fine-tuning artificial cytochrome b5 proteins and designing new, versatile variants., (© 2025 Wiley-VCH GmbH.)

Cytochrome b5 (CB5) is a small heme-binding protein, known as an electron donor delivering reducing power to the terminal enzymes involved in oxidative reactions. In plants, the CB5 protein family is substantially expanded both in its isoform numbers and cellular functions, compared to its yeast and mammalian counterparts. As an electron carrier, plant CB5 proteins function not only in fatty acid desaturation, hydroxylation and elongation, but also in the formation of specialized metabolites such as flavonoids, phenolic esters, and heteropolymer lignin. Furthermore, plant CB5s are found to interact with different non-catalytic proteins such as ethylene signaling regulator, cell death inhibitor, and sugar transporters, implicating their versatile regulatory roles in coordinating different metabolic and cellular processes, presumably in respect to the cellular redox status and/or carbon availability. Compared to the plentiful studies on biochemistry and cellular functions of mammalian CB5 proteins, the cellular and metabolic roles of plant CB5 proteins have received far less attention. This article summarizes the fragmentary information pertaining to the discovery of plant CB5 proteins, and discusses the conventional and peculiar functions that plant CB5s might play in different metabolic and cellular processes. Gaining comprehensive insight into the biological functions of CB5 proteins could offer effective biotechnological solutions to tailor plant chemodiversity and cellular responses to environment stimuli. [ABSTRACT FROM AUTHOR]

We recently reported that the membrane‐associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5) domain proteins, called cytb5M (MAPR‐like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme‐binding orientation at an approximately 90° rotation relative to classical cytb5, as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the crystal structure of an archaeal cytb5M domain (Methanococcoides burtoniiWP_011499504.1, PDB:6VZ6). It exhibits similar heme binding to the 6NZX cytb5M, supporting the deduction that MAPR‐like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene. [ABSTRACT FROM AUTHOR]

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment. [ABSTRACT FROM AUTHOR]

Cytochrome b5 (CB5) is a small heme-binding protein, known as an electron donor delivering reducing power to the terminal enzymes involved in oxidative reactions. In plants, the CB5 protein family is substantially expanded both in its isoform numbers and cellular functions, compared to its yeast and mammalian counterparts. As an electron carrier, plant CB5 proteins function not only in fatty acid desaturation, hydroxylation and elongation, but also in the formation of specialized metabolites such as flavonoids, phenolic esters, and heteropolymer lignin. Furthermore, plant CB5s are found to interact with different non-catalytic proteins such as ethylene signaling regulator, cell death inhibitor, and sugar transporters, implicating their versatile regulatory roles in coordinating different metabolic and cellular processes, presumably in respect to the cellular redox status and/or carbon availability. Compared to the plentiful studies on biochemistry and cellular functions of mammalian CB5 proteins, the cellular and metabolic roles of plant CB5 proteins have received far less attention. This article summarizes the fragmentary information pertaining to the discovery of plant CB5 proteins, and discusses the conventional and peculiar functions that plant CB5s might play in different metabolic and cellular processes. Gaining comprehensive insight into the biological functions of CB5 proteins could offer effective biotechnological solutions to tailor plant chemodiversity and cellular responses to environment stimuli.

Cytochrome b5 (Cyt-b5) is a small heme protein and known to be involved in a wide range of biochemical transformations, including cytochrome P450 monooxygenase (CYP)-mediated metabolism of endogenous and exogenous compounds. Studies on Cyt-b5 are more concentrated in mammals, but are relatively rare in insects. The characteristics and function of Cyt-b5 from Locusta migratoria have not been described yet. We sequenced the full-length cDNA sequence of Cyt-b5 from L. migratoria (LmCyt-b5) by reverse transcription-PCR (RT-PCR) based on locust transcriptome database. The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea. LmCyt-b5 was highly expressed in ovary, Malpighian tubules, midgut, gastric caeca, and fat bodies. Silencing of LmCyt-b5 had no effect on the susceptibility of L. migratoria to four different insecticides. Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin (7-EC). However, coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of LmCYP6FD1 with cytochrome P450 reductase (LmCPR) or LmCyt-b5 separately. These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments. Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L. migratoria.

Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome b5 (CYT b5) production in Escherichia coli N4830-1, as the heterologous host. Cyt b5 was introduced as a plasmid with between 0 and 6 copies under a strong promoter. The FT-IR spectroscopy results combined with multivariate chemometric analysis illustrated discriminations among culture conditions as well as revealing features that correlated to the different cytb5 gene copy numbers. The second derivative of the FT-IR spectral data allowed for the quantitative detection of Cyt b5 directly inside the intact cells without the need for extraction, and highlighted changes in protein secondary structure that was directly correlated to the cytb5 gene copy number and protein content, and was in complete agreement with quantitative findings of standard traditional techniques such as SDS–PAGE and western blot analysis.

Cytochrome b5 is a small electron transport protein that is found in animals, plants, fungi and photosynthetic proteobacteria where it plays key metabolic roles in energy production, lipid and sterol biosynthesis and cytochrome P450 biochemistry. Previously it was shown that a gene encoding a soluble and functional cytochrome b5 protein was encoded in the large double stranded DNA virus OtV2 that infects the unicellular marine green alga Ostreococcus tauri , the smallest free-living eukaryote described to-date. This single gene represented a unique finding in the virosphere. We now report that genes for soluble and membrane-bound cytochromes b5 also occur in giant viruses in the proposed order Megavirales, particularly the AT-rich Mimiviridae and Tupanviruses . Conversely, other members of the Megavirales taxa such as the GC-rich Pandoraviridae have not been found to encode cytochrome b5 as yet. Megaviruses encoding cytochrome b5 have been isolated from the deep ocean, from freshwater and terrestrial sources, as well as from human patients. Giant virus cytochrome b5 proteins share high sequence identity with one another (45-95% depending on group) but no more than 25% identity with the cytochrome b5 gene product we identified in Acanthamoeba castellanii , an amoeba host for many giant viruses. Thus, the origin of the unique cytochrome b5 genes in giant viruses remains unknown. Examination of viral cytochrome b5 primary amino acid sequences revealed that some have either a N- or C-terminal transmembrane anchor, whilst others lack a membrane anchor and are thus predicted to be soluble proteins. This cytochrome b5 topography suggests adapted biochemical functions in those viruses. Our findings raise questions regarding the evolution and diversity of cytochrome b5 proteins in nature, adding to questions about the origin of viral haemoproteins in general., Competing Interests: Additional Declarations: No competing interests reported.

Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease. • Cytoglobin controls nitric oxide (NO) metabolism through NO dioxygenation. • Tobacco smoking exposure induces loss of vascular NO; however, the cause was unknown. • Vascular smooth muscle cell exposure first triggered increased superoxide generation. • This was followed by induction of cytoglobin and the B5/B5R reducing system. • Together, this greatly increased the rate of NO decay in vascular smooth muscle cells. [ABSTRACT FROM AUTHOR]

Potassium (K+) is an essential element for growth and development in both animals and plants, while high levels of environmental sodium (Na+) represent a threat to most plants. The uptake of K+ from high-saline environments is an essential mechanism to maintain intracellular K+/Na+ homeostasis, which can help reduce toxicity caused by Na+ accumulation, thereby improving the salt tolerance of plants. However, the mechanisms and regulation of K+-uptake during salt stress remain poorly understood. In this study, we identified an endoplasmic reticulum–localized cytochrome b5 (OsCYB5-2) that interacted with a high-affinity K+ transporter (OsHAK21) at the plasma membrane. The association of OsCYB5-2 with the OsHAK21 transporter caused an increase in transporter activity by enhancing the apparent affinity for K+- binding but not Na+-binding. Heme binding to OsCYB5-2 was essential for the regulation of OsHAK21. High salinity directly triggered the OsHAK21–OsCYB5-2 interaction, promoting OsHAK21- mediated K+-uptake and restricting Na+ entry into cells; this maintained intracellular K+/Na+ homeostasis in rice cells. Finally, overexpression of OsCYB5-2 increased OsHAK21-mediated K+ transport and improved salt tolerance in rice seedlings. This study revealed a posttranslational regulatory mechanism for HAK transporter activity mediated by a cytochrome b5 and highlighted the coordinated action of two proteins to perceive Na+ in response to salt stress. [ABSTRACT FROM AUTHOR]

Soybean mosaic virus (SMV) is a potyvirus found worldwide in soybean (Glycine max). GmCYB5-4 is a strong candidate interactor of P3. In this study, we comprehensively analyzed the GmCYB5 family in soybeans, including its distribution on chromosomes, promoter analysis, conserved motifs, phylogenetic analysis, and expression patterns. We cloned the full-length GmCYB5-4 and examined its interaction with P3 in yeast, which was later confirmed using bimolecular fluorescence complementation (BiFc). We silenced GmCYB5-4 using a bean pottle mosaic viris (BPMV) based system to generate Sil CYB5-4 tissues, which surprisingly knocked down four isoforms of GmCYB5s for functional characterization. Sil CYB5-4 plants were challenged with the SC3 strain to determine its involvement in SMV infection. Silencing GmCYB5-4 increased SMV accumulation, indicating that GmCYB5-4 inhibited SMV proliferation. However, further experiments are needed to elucidate the mechanism underlying the involvement of GmCYB5-4 in SMV infection. • GmCYB5s were comprehensive analyzed at the genome-wide level in soybean. • GmCYB5-4 interacts with P3 in yeast and tobacco. • The silencing tissue Sil CYB5-4 knocked down four GmCYB5s expression. • The Sil CYB5-4 plant accumulated more SMV as compared to V plant. [ABSTRACT FROM AUTHOR]

Cyanobacteria are prolific producers of natural products, including polyketides and hybrid compounds thereof. Type III polyketide synthases (PKSs) are of particular interest, due to their wide substrate specificity and simple reaction mechanism, compared with both type I and type II PKSs. Surprisingly, only two type III PKS products, hierridins, and (7.7)paracyclophanes, have been isolated from cyanobacteria. Here, we report the mining of 517 cyanobacterial genomes for type III PKS biosynthesis gene clusters. Approximately 17% of the genomes analyzed encoded one or more type III PKSs. Together with already characterized type III PKSs, the phylogeny of this group of enzymes was investigated. Our analysis showed that type III PKSs in cyanobacteria evolved into three major lineages, including enzymes associated with 1) (7.7)paracyclophane-like biosynthesis gene clusters, 2) hierridin-like biosynthesis gene clusters, and 3) cytochrome b5 genes. The evolutionary history of these enzymes is complex, with some sequences partitioning primarily according to speciation and others putatively according to their reaction type. Protein modeling showed that cyanobacterial type III PKSs generally have a smaller active site cavity (mean = 109.035 Å3) compared with enzymes from other organisms. The size of the active site did not correlate well with substrate size, however, the "Gatekeeper" amino acid residues within the active site were strongly correlated to enzyme phylogeny. Our study provides unprecedented insight into the distribution, diversity, and molecular evolution of cyanobacterial type III PKSs, which could facilitate the discovery, characterization, and exploitation of novel enzymes, biochemical pathways, and specialized metabolites from this biosynthetically talented clade of microorganisms. [ABSTRACT FROM AUTHOR]

The electron donor cytochrome b5 (CybE/Cyb5) fuels the activity of the ergosterol biosynthesis-related P450 enzymes (P450s) by providing electrons to P450s to promote ergosterol biosynthesis. Previous studies reported that a lack of Aspergillus fumigatus CybE reduces the proportion of ergosterol in total sterols and induces severe growth defects. However, the molecular characteristics of CybE and the underlying mechanism for CybE maintaining A. fumigatus growth remain poorly understood. Here, we found that the C terminus of CybE, with two transmembrane domains, is located at the endoplasmic reticulum. Therefore, a strain lacking the C terminus of CybE shows a defective growth phenotype similar to that of a cybE deletion strain. Notably, cybE deletion reduced the accumulation of the sterol-rich plasma membrane domains (SRDs; the assembly platform of polarity factors/cell end markers and growth machinery) in hyphal tips and decreased membrane fluidity, which corresponds to tardiness of hyphal extension and hypersensitivity to low temperature in the cybE deletion mutant. Additionally, overexpressing another electron donor, a heme-independent P450 reductase (cytochrome P450 reductase [CPR]), significantly rescued growth defects and recovered SRD accumulation in the cybE deletion mutant almost to the wild-type level, suggesting that CybE maintaining the growth and deposition of SRDs in hyphal tips is attributed to its nature as an electron donor. Protein pulldown assays revealed that CybE probably participates in the metabolism and transfer of lipids, construction of cytoskeleton, and mitochondria-associated energy metabolism to maintain the SRD accumulation in hyphal tips, membrane fluidity, and hyphal extension. The findings in this study give a hint that inhibition of CybE may be an effective strategy for resisting the infection of the human pathogen A. fumigatus.IMPORTANCE Investigating the knowledge of the growth regulation in the human opportunistic pathogen Aspergillus fumigatus is conducive to designing a new antifungal approach. The electron donor cytochrome b5 (CybE) plays a crucial role in maintaining the normal growth of A. fumigatus; however, the potential mechanism remains elusive. Here, we characterized the molecular features of CybE and found that the C terminus with two transmembrane domains is required for its endoplasmic reticulum (ER) localization and functions. In addition, we demonstrated that CprA, an electron donor-heme-independent P450 reductase, provides a reciprocal function for the missing cytochrome b5 protein-CybE in A. fumigatus. CybE maintains the normal growth probably via supporting two crucial physiological processes, the sterol-rich plasma membrane domain (SRD) accumulation in hyphal tips and membrane fluidity. Therefore, our findings reveal the mechanisms underlying the regulatory effect of CybE on A. fumigatus growth and indicate that inhibition of CybE might be an effective approach for alleviating A. fumigatus infection. [ABSTRACT FROM AUTHOR]

[ Objectives] The purpose of this study was to clarify the optimum length of the chromogenic fragments of Cyt-b5 containing Cyt-b5 -like heme/ steroid binding domain (Cyt-b5D) and its adaptability using different prokaryotic expression vectors, and screen out effective visual tag in order to improve the efficiency and convenience of vector construction. [ Methods] The structural domain of Cyt-h5 protein of Chilo suppressalis was analysed and divided into different fragments with different sizes, such as OA, OB, OC, etc. containing Cyt-b5D. These fragments were ligated with prokaryotic expression vectors, including pET-28a ( +), pET-30a ( +) and pET-4la( +),respectively and expressed in Escherichia coli. The color reaction and absorbance of bacterial solution of the screened chromogenic fragments co-expressed with the C. suppressalis RDLJ were detected at different time points. The recombinant proteins expressed by the chromogenic fragments and co-expressed with RDLJ were purified by affinity chromatography and detected by the Western blot. [Results ] The OA fragment was the best chromogenic fragment, which could express the red proteins using three prokaryotic expression vectors. In addition, its color visualization was stable and adaptable when co-expressed with RDLJ. The growth status of chromogenic bacterial solution could he real-time monitored on the basis of its characteristic absorption at 420 nm, whereas the process of protein purification could he estimated according to the red change of the chromatographic column. In addition, the fusion protein could he detected by the Western blot, suggesting that the presence of exogenous chromogenic fragment did not disturb the accurate identification of the target protein. [ Conclusions ] The chromogenic fragment ( OA) could he used as a visual tag in prokaryotic expression system, he able to provide the indication tag for protein expression and purification, and further improve the convenience and timeliness of the experimental operations as described above. [ABSTRACT FROM AUTHOR]

Cytochrome b5 is an essential electron transfer protein, which is ubiquitously found in living systems and involved in wide variety of biological processes. Tardigrades (also known as water bears), some of which are famous for desiccation resistance, have many proteins unique to them. Here, we report spectroscopic and structural characterization of a cytochrome b5 like protein from one of the desiccation‐tolerant tardigrades, Ramazzottius varieornatus strain YOKOZUNA‐1 (RvCytb5). A 1.4 Å resolution crystal structure revealed that RvCytb5 is a new cytochrome b5 protein specific to tardigrades. PDB Code(s): 7BWH; [ABSTRACT FROM AUTHOR]

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo‐ and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone‐precursor premedrol by optimizing the CYP21A2‐based whole‐cell system on a laboratory scale. We successfully improved the whole‐cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N‐terminally truncated version of the bovine NADPH‐dependent cytochrome P450 reductase (bCPR−27) and coexpression of microsomal cytochrome b5; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed‐batch protocol. By overcoming the presented limitations in whole‐cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L−1·d−1 premedrol. [ABSTRACT FROM AUTHOR]

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of modern protein interactomics. This study is a logical continuation of the cycle of our previous works aimed at the PPIs between components of the cytochrome P450-dependent monooxygenase system. Using an optical biosensor employing surface plasmon resonance (SPR biosensor), a comparative analysis characterizing kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 and cytochrome P450s was performed using two different protocols for protein immobilization: (1) covalent non-oriented immobilization on the carboxymethyl dextran chip and (2) non-covalent oriented immobilization in the lipid environment. In the case of the second protocol, PPIs were characterized by 2.5-fold higher affinity due to a decrease in rate dissociation constants values of the studied complexes. [ABSTRACT FROM AUTHOR]

NADH cytochrome b 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b 5 (b 5 ) and cytochrome b 5 reductase (b 5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b 5 domain and a construct containing the CS and b 5 R domains (CS/b 5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b 5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11-residue α-helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in the b 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N-terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochrome b 5 proteins from three kingdoms among eukaryotes., (© 2023 Wiley Periodicals LLC.)

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.

Recently, we observed that at extreme alkaline pH, cytochrome b5 (Cb5) acquires a peroxidase-like activity upon formation of a low spin hemichrome associated with a non-native state. A functional characterization of Cb5, in a wide pH range, shows that oxygenase/peroxidase activities are stimulated in alkaline media, and a correlation between tyrosine ionization and the attained enzymatic activities was noticed, associated with an altered heme spin state, when compared to acidic pH values at which the heme group is released. In these conditions, a competitive assay between imidazole binding and Cb5 endogenous heme ligands revealed the appearance of a binding site for this exogenous ligand that promotes a heme group exposure to the solvent upon ligation. Our results shed light on the mechanism behind Cb5 oxygenase/peroxidase activity stimulation in alkaline media and reveal a role of tyrosinate anion enhancing Cb5 enzymatic activities on the distorted protein before maximum protein unfolding. [ABSTRACT FROM AUTHOR]

Herein, the in vitro metabolism of tyrosine kinase inhibitor cabozantinib, the drug used for the treatment of metastatic medullary thyroid cancer and advanced renal cell carcinoma, was studied using hepatic microsomal samples of different human donors, human recombinant cytochromes P450 (CYPs), flavin-containing mono-oxygenases (FMOs) and aldehyde oxidase. After incubation with human microsomes, three metabolites, namely cabozantinib N-oxide, desmethyl cabozantinib and monohydroxy cabozantinib, were detected. Significant correlations were found between CYP3A4 activity and generation of all metabolites. The privileged role of CYP3A4 was further confirmed by examining the effect of CYP inhibitors and by human recombinant enzymes. Only four of all tested human recombinant cytochrome P450 were able to oxidize cabozantinib, and CYP3A4 exhibited the most efficient activity. Importantly, cytochrome b5 (cyt b5) stimulates the CYP3A4-catalyzed formation of cabozantinib metabolites. In addition, cyt b5 also stimulates the activity of CYP3A5, whereas two other enzymes, CYP1A1 and 1B1, were not affected by cyt b5. Since CYP3A4 exhibits high expression in the human liver and was found to be the most efficient enzyme in cabozantinib oxidation, we examined the kinetics of this oxidation. The present study provides substantial insights into the metabolism of cabozantinib and brings novel findings related to cabozantinib pharmacokinetics towards possible utilization in personalized medicine.

Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b 5 —are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

Highlights • Affinity of CYPs with both CPR and Adx interactions did not affected by compounds. • Affinity of CYB5A/CYPs interactions specifically modulated by steroidal substrates. • Affinity modulation of CYP/CYB5A complexes was in a multidirectional mode. Abstract The goal of this work was to test the hypothesis that the affinity of protein-protein interactions in the cytochrome P450-dependent monooxygenase system is modulated by the low-molecular-weight compounds (substrates or inhibitors). The surface plasmon resonance (SPR) based study was carried out using the recombinant protein preparations of three microsomal cytochromes P450 (CYP17A1, CYP21A2, and CYP2C19) and their redox partners: cytochrome b5 (CYB5A), NADPH - cytochrome P450 reductase (CPR), and also iron-sulfur protein adrenodoxin (Adx). As a result, we have revealed some specificity of the influence of the steroid substrates on the binding affinity of CYPs with their redox partners, namely: the lack of effect on CPR/CYPs and Adx/CYP complex formation, and a significant effect on interactions between CYB5A and steroidogenic CYPs. The equilibrium dissociation constant (Kd) value of the CYB5A/CYP17A1 complex decreased by 5 times in the presence of progesterone (P4), which was due to a 10 times increase in the association rate constant (k on). In this case, a twofold increase in the dissociation rate constant (k off) value of CYB5A/CYP17A1 complex formation was observed. It was also demonstrated that the affinity of CYB5A/CYP17A1 interaction increased in the presence of two other steroidal substrates 17α-hydroxyprogesterone and pregnenolone and that effect was comparable with P4. In contrast, only the twofold decrease in the affinity of CYB5A/CYP21A2 interaction in the presence of P4 was caused by a slight increase in the k off value (the k on value of the complex did not change). This indicates a different format of the steroidal substrates effects expressed in a change in the stability of the CYB5A/CYPs complexes. Thus, it was found that P4 modulated the both kinetic and equilibrium constants of CYB5A/CYP17A1 and CYB5/CYP21A2 complex formation and complexes, while not affecting the CYB5A/CYP2C19 interaction (2C19 is the cytochrome P450 isoenzyme possessing broad substrate specificity), thereby indicating a specific influence of steroidal substrates on interactions involving steroidogenic CYPs. Our results are consistent with current understanding of the role of CYB5A as a regulator of cytochrome P450 activity in P450-dependent monooxygenase system. [ABSTRACT FROM AUTHOR]

Abstract The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b 5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b 5 /P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro , but not in vivo. Highlights • The anticancer drug ellipticine is activated by cytochrome P450 (CYP) enzymes. • Cytochrome b 5 (Cyb5) and NADPH:CYP oxidoreductase (POR) modulate CYP enzyme activity. • Ellipticine bioactivation was investigated in mouse lines with hepatic deletions of Cyb5 and/or POR. • POR strongly contributed to ellipticine bioactivation in vivo. • Cyb5 contributed little to ellipticine bioactivation in vivo contrasting previous in vitro studies. [ABSTRACT FROM AUTHOR]

Purpose: This work is performed to determine if there is a potential for the accumulation and potential toxicity of triacetone triperoxide (TATP) in canines. Additional utility of this information may suggest human toxicity and possibly detection of biomarkers, metabolites or intact molecule of those using this material for nefarious reasons.Methods: Liquid chromatography/mass spectrometry of dog liver microsome (DLM) incubation samples of TATP was used to measure substrate depletion. Trapping of electrophilic products was performed using glutathione (GSH) and semicarbazide. Comparisons were made to free hydroperoxides found in methyl ethyl ketone peroxides (MEKP).Results: The non-specific Km value of 2.2 μM and a Vmax of 1.1 nmol/min/mg of protein were determined. Canine recombinant cytochrome P450 (rCYP) 2B11 with human cytochrome b5 was found to catalyze the NADPH-dependent metabolism of TATP into its only phase I metabolite, hydroxy-TATP (TATP-OH). No secondary metabolite(s) or degraded products were detected or trapped from microsomal incubations. MEKP subjected to similar conditions was found to undergo significant metabolism, semicarbazide trapping and rapid oxidation of GSH to GSSG. The synthesized TATP-OH metabolite incubated in DLM progressed three times faster than TATP metabolism with no secondary metabolites found or trapped.Conclusions: TATP does not react as MEKP suggesting that TATP does not ring-open to form hydroperoxides. TATP and TATP-OH compete for the same enzyme, with TATP dominating this competition. Failure to detect additional metabolite(s) suggests they may be too small to detect by our system or bound covalently to a protein or polymer in the incubation reaction. [ABSTRACT FROM AUTHOR]

Abstract NADH cytochrome b 5 reductase mediates electron transfer from NADH to cytochrome b 5 utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b 5 is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b 5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5–10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b 5 by cytochrome b 5 reductase, as measured by heme spectral changes in cytochrome b 5. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b 5. Chemical redox cycling by cytochrome b 5 reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b 5 reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b 5 , are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals. Graphical abstract Unlabelled Image Highlights • Cytochrome b 5 reductase (cypb5) mediates chemical redox cycling. • Chemical redox cycling by cypb5 generates reactive oxygen species. • Redox cycling by cypb5 is NADH- and oxygen-dependent. • Redox cycling inhibits electron transfer from cypb5 to cytochrome b 5. [ABSTRACT FROM AUTHOR]

Cytochrome P450 17A1 (CYP17A1) catalyzes the synthesis of androgens from the steroid precursors pregnenolone and progesterone in a two‐step reaction process: allylic hydroxylation and carbo‐carbon bond scission. Cytochrome b5 (Cyt‐b5) is a stimulator of the second lyase reaction, but the chemical mechanism is unclear. We have shown previously that this stimulatory effect requires redox active Cyt‐b5. To investigate the origin of the lyase reaction enhancement by electron transfer from Cyt‐b5, we measured the reduction rates of oxy‐ferrous substrate‐bound CYP17A1 by Cyt‐b5 and by cytochrome P450 reductase (CPR) coincorporated in Nanodiscs using stopped flow spectroscopy. We observed that Cyt‐b5 reduces oxy‐ferrous CYP17A1 10‐fold faster than CPR, with the rate similar to that observed in a ternary complex of all three proteins. [ABSTRACT FROM AUTHOR]

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis. [ABSTRACT FROM AUTHOR]

Abstract: CYP725A4 is a P450 enzyme from Taxus cuspidata that catalyzes the formation of taxadiene‐5α‐ol (T5α‐ol) from taxadiene in paclitaxel biosynthesis. Past attempts expressing CYP725A4 in heterologous hosts reported the formation of 5(12)‐oxa‐3(11)‐cyclotaxane (OCT) and/or 5(11)‐oxa‐3(11)‐cyclotaxane (iso‐OCT) instead of, or in addition to, T5α‐ol. Here, we report that T5α‐ol is produced as a minor product by Escherichia coli expressing both taxadiene synthase and CYP725A4. The major products were OCT and iso‐OCT, while trace amounts of unidentified monooxygenated taxanes were also detected by gas chromatography–mass spectrometry. Since OCT and iso‐OCT had not been found in nature, we tested the hypothesis that protein–protein interaction of CYP725A4 with redox partners, such as cytochrome P450 reductase (CPR) and cytochrome b5, may affect the products formed by CYP725A4, possibly favoring the formation of T5α‐ol over OCT and iso‐OCT. Our results show that coexpression of CYP725A4 with CPR from different organisms did not change the relative ratios of OCT, iso‐OCT, and T5α‐ol, while cytochrome b5 decreased overall levels of the products formed. Although unsuccessful in finding conditions that promote T5α‐ol formation over other products, we used our results to clarify conflicting claims in the literature and discuss other possible approaches to produce paclitaxel via metabolic and enzyme engineering. [ABSTRACT FROM AUTHOR]

In recent years, a growing appreciation has developed for the importance of protein-protein interactions to modulate the function of drug metabolizing enzymes. Accompanied with this appreciation, new methods and technologies have been designed for analyzing protein-protein interactions both in vitro and in vivo. These technologies have been applied to several classes of drug metabolizing enzymes, including: cytochrome P450's (CYPs), monoamine oxidases (MAOs), UDP-glucuronosyltransferases (UGTs), glutathione S-transferases (GSTs), and sulfotransferases (SULTs). In this review, we offer a brief description and assessment of the impact of many of these technologies to the study of protein-protein interactions in drug disposition. The still expanding list of these techniques and assays has the potential to revolutionize our understanding of how these enzymes carry out their important functions in vivo.

Soybean mosaic virus (SMV) is one of the most destructive viral diseases in soybeans (Glycine max). In this study, an interaction between the SMV P3 protein and cytochrome b5 was detected by yeast two-hybrid assay, and bimolecular fluorescence complementation assay showed that the interaction took place at the cell periphery. Further, the interaction was confirmed by co-immunoprecipitation analysis. Quantitative real-time polymerase chain reaction analysis revealed that GmCYB5 gene was differentially expressed in resistant and susceptible soybean plants after inoculation with SMV-SC15 strain. To test the involvement of this gene in SMV resistance, the GmCYB5 was silenced using a bean pod mottle virus (BPMV)-based vector construct. Results showed that GmCYB5-1 was 83% and 99% downregulated in susceptible (NN1138-2) and resistant (RN-9) cultivars, respectively, compared to the empty vector-treated plants. Silencing of GmCYB5 gene promotes SMV replication in soybean plants. Our results suggest that during SMV infection, the host CYB5 protein targets P3 protein to inhibit its proliferation. Taken together, these results suggest that CYB5 is an important factor in SMV infection and replication in soybeans, which could help soybean breeders develop SMV resistant soybean cultivars.

Tail‐anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein‐free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5‐ER and b5‐RR, which differ only in the charge of the C‐terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell‐free and in cellula results, we analyzed targeting in digitonin‐permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C‐terminal negative charge of b5‐ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI) reduced insertion of b5‐ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI, Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C‐terminal charge, and reveal a novel, substrate‐specific ER‐targeting pathway. [ABSTRACT FROM AUTHOR]

The activity of liver components of monooxygenase system (MOS) have been investigated in rats with tumor under conditions of ω-3 polyunsaturated fatty acids (PUFAs) administration. It was shown, that the intensive growth period of Guerin's carcinoma is thus associated with cytochrome b5-reductase activity increased 2 times, and cytochrome b5 content decreased 2.8 times, and increased O2- generation rate in reductive chain of MOS in comparison to control. We found NADPH-cytochrome P450-reductase activity decreased 2,4 times and cytochrome P450 content decreased 2,8 times in oxygenative chain of MOS of rats with transplanted Guerin's carcinoma in the logarithmic phases of oncogenesis as compared with the control. The decrease of cytochrome P450 level in the liver microsomal fraction in the logarithmic phases of oncogenesis may be associated with its transition into an inactive form - cytochrome P420. ω-3 PUFAs (eicosapentaenoic acid and docosahexaenoic acid) administration before and after transplantation of Guerin's carcinoma resulted in the decrease of NADH-cytochrome b5-reductase activity, increase of cytochrome b5 level, NADРH-cytochrome Р450 reductase activity and cytochrome P450 level in the liver microsomal fraction in the logarithmic phases of oncogenesis as compared with the tumor-bearing rats. We have established the correction of reductive and oxygenative chain of MOS under ω-3 PUFAs administration prior Guerin's carcinoma injection as compared with the tumor-bearing rats. Yet the changes are less pronounced than those of the group receiving ω-3 PUFAs both prior to and post-Guerin's carcinoma implantation. ω-3 PUFAs administration post-Guerin's carcinoma injection does not influence on activity of liver MOS components of tumor-bearing rats. [ABSTRACT FROM AUTHOR]

In flow-based systems, polymer pipes, pipette tips or capillaries are used for the delivery of analyte solution into the measuring cell. In such systems, a generation of charge in the solution occurs [1,2]. We have performed monitoring of charge accumulation for water and 10 −15 M cytochrome b5 solution. A non-linear time dependence of the charge accumulation has been observed. A phenomenon of occurrence of stepwise changes in the charge value has been registered. This effect must be taken into account in systems, where the charge state of the protein molecules has a significant impact on the detection efficiency. [ABSTRACT FROM AUTHOR]

In recent years, a growing appreciation has developed for the importance of protein-protein interactions to modulate the function of drug metabolizing enzymes. Accompanied with this appreciation, new methods and technologies have been designed for analyzing protein-protein interactions both in vitro and in vivo. These technologies have been applied to several classes of drug metabolizing enzymes, including: cytochrome P450's (CYPs), monoamine oxidases (MAOs), UDP-glucuronosyltransferases (UGTs), glutathione S-transferases (GSTs), and sulfotransferases (SULTs). In this review, we offer a brief description and assessment of the impact of many of these technologies to the study of protein-protein interactions in drug disposition. The still expanding list of these techniques and assays has the potential to revolutionize our understanding of how these enzymes carry out their important functions in vivo. [ABSTRACT FROM AUTHOR]

Omega-7 (ω-7) fatty acids have potential application in the fields of nutraceutical, agricultural, and food industry. The natural ω-7 fatty acids are currently from plants or vegetable oils, which are unsustainable and limited by the availability of plant sources. Here, we developed an innovative biosynthetic route to produce vaccenic acid (C18:1 ω-7) while minimizing oleic acid (C18:1 ω-9) content in Saccharomyces cerevisiae . We have engineered S. cerevisiae to produce C18:1 ω-7 by expressing a fatty acid elongase from Rattus norvegicus . To reduce the content of C18:1 ω-9, the endogenous desaturase Ole1 was replaced by the desaturase, which has specific activity on palmitoyl-coenzyme A (C16:0-CoA). Finally, the production of free C18:1 ω-7 was improved by optimizing the source of cytochrome b 5 and overexpressing endoplasmic reticulum chaperones. After combining these strategies, the yield of C18:1 ω-7 was increased from 0 to 9.3 mg/g DCW and C18:1 ω-9 was decreased from 25.2 mg/g DCW to 1.6 mg/g DCW. This work shows a de novo synthetic pathway to produce the highest amount of free C18:1 ω-7 with a low content of C18:1 ω-9 in S. cerevisiae .