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Mining Cytochrome b561 proteins from plant genomes.

ABSTRACT: Cytochrome b561 (Cyt-b561) proteins are important for plant growth, development, and prevention of damage to plants. Because of their high sequence divergence, thorough mining of Cyt-b561 proteins from plant genomes are not easy. Currently there is only one Cyt-b561 gene found in the maize and none in the soybean genome. However, 22 have been identified in the Arabidopsis thaliana genome. We tested alignment-free protein classifiers based on partial least squares (PLS) and support vector machines to identify Cyt-b561. These classifiers performed better than profile hidden Markov models and PSI-BLAST. Using these classifiers we identified new Cyt-b561-related proteins from four plant genomes.

SUBMITTER: Opiyo SO

PROVIDER: S-EPMC2846449 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Mining Cytochrome b561 proteins from plant genomes.

Opiyo Stephen O SO Moriyama Etsuko N EN

International journal of bioinformatics research and applications 20100101 2

Cytochrome b561 (Cyt-b561) proteins are important for plant growth, development, and prevention of damage to plants. Because of their high sequence divergence, thorough mining of Cyt-b561 proteins from plant genomes are not easy. Currently there is only one Cyt-b561 gene found in the maize and none in the soybean genome. However, 22 have been identified in the Arabidopsis thaliana genome. We tested alignment-free protein classifiers based on partial least squares (PLS) and support vector machine ...[more]

PMID: 20223741

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Project description:Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, we carried out a bioinformatics-based investigation of cytb561 family members from nine species representing eight insect orders. We performed a phylogenetic analysis to classify insect cytb561 ortholog groups. We then conducted sequence analyses and analyzed protein models to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. Our study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. We found that CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Our investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved "KXXXXKXH" non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects.

Project description:Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (? 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the ?-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) ?-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.

Dataset Information

Mining Cytochrome b561 proteins from plant genomes.

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Mining Cytochrome b561 proteins from plant genomes.

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