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Cloning and characterization of a maize cytochrome-b5 reductase with Fe3+-chelate reduction capability.

ABSTRACT: We previously purified an NADH-dependent Fe3+-chelate reductase (NFR) from maize roots with biochemical features of a cytochrome-b5 reductase (b5R) [Sparla, Bagnaresi, Scagliarini and Trost (1997) FEBS Lett. 414, 571-575]. We have now cloned a maize root cDNA that, on the basis of sequence information, calculated parameters and functional assay, codes for NFR. Maize NFR has 66% and 65% similarity to mammal and yeast b5R respectively. It has a deduced molecular mass of 31.17 kDa and a pI of 8.53. An uncharged region is observed at its N-terminus but no myristoylation consensus site is present. Taken together, these results, coupled with previous biochemical evidence, prove that NFR belongs to the b5R class and document b5R from a plant at the molecular level for the first time. We have also identified a putative Arabidopsis thaliana NFR gene. Its organization (nine exons) closely resembles mammalian b5Rs. Several NFR isoforms are expected to exist in maize. They are probably not produced by alternative translational mechanisms as occur in mammals, because of specific constraints observed in the maize NFR cDNA sequence. In contrast with yeast and mammals, tissue-specific and various subcellular localizations of maize b5R isoforms could result from differential expression of the various members of a multigene family. The first molecular characterization of a plant b5R indicates an overall remarkable evolutionary conservation for these versatile reductase systems. In addition, the well-characterized Fe3+-chelate reduction capabilities of NFR, in addition to known Fe3+-haemoglobin reduction roles for mammal b5R isoforms, suggest further and more generalized roles for the b5R class in endocellular iron reduction.

SUBMITTER: Bagnaresi P

PROVIDER: S-EPMC1220078 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

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Project description:Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins might eventually transfer electrons to oxygen to form reactive oxygen species. Additionally, an inefficient electron transfer to redox acceptors can lead to electron uncoupling and superoxide anion formation by the reductase. Many efforts have been made to characterize the involved catalytic domains in the electron transfer from the reduced flavoprotein to its electron acceptors, such as cytochrome b5, through a detailed description of the flavin and NADH-binding sites. This information might help to understand better the processes and modifications involved in reactive oxygen formation by the cytochrome b5 reductase. Nevertheless, more than half a century since this enzyme was first purified, the one-electron transfer process toward potential electron acceptors of the reductase is still only partially understood. New advances in computational analysis of protein structures allow predicting the intramolecular protein dynamics, identifying potential functional sites, or evaluating the effects of microenvironment changes in protein structure and dynamics. We applied this approach to characterize further the roles of amino acid domains within cytochrome b5 reductase structure, part of the catalytic domain, and several sensors and structural domains involved in the interactions with cytochrome b5 and other electron acceptors. The computational analysis results allowed us to rationalize some of the available spectroscopic data regarding ligand-induced conformational changes leading to an increase in the flavin adenine dinucleotide (FAD) solvent-exposed surface, which has been previously correlated with the formation of complexes with electron acceptors.

Project description:Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of arylhydroxylamine carcinogens, and we further hypothesize that this pathway may be a source of individual variability with respect to cancer susceptibility following 4-ABP or PhIP exposure.

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Cloning and characterization of a maize cytochrome-b5 reductase with Fe3+-chelate reduction capability.

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