338 research outputs found
Dynamics of Electron Transport in Cytochrome P450 Systems Studied at Sub-Zero Temperature
Experimentation in fluid mixed solvents (1 : 1 v/v phosphate
buffer ethylene glycol) at sub-zero temperatures has permitted us
to record the two univalent reductions of the bacterial cytochrome
P450 by the natural electron donor putidaredoxin, without recycling
or alternative pathway reactions. Dynamic evidence shows the formation of putidaredoxincytochrome complexes prior to electron
transfer. The complex formation is rate limiting in the first reduction
and in our experimental conditions. The kinetics of binding
between the two oxidized proteins has also been recorded in the
same medium under various conditions of concentration, temperature
and ionic strength. At very low ionic strength, the rate is
limited by electrostatic repulsion between the two negatively charge
proteins; above I = 0.03 this effect appears negligible and the affinity
seems to be governed by hydrophobic interaction free energy
Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase
The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family. We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777. The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination. These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle
Electronic Structure of Cytochrome P450
The optical properties of P450 have been investigated by means
of polarized absorption spectroscopy of single crystals of camphor-
bound P450CAM in the oxidized, reduced, and CO-reduced states,
and iterative extended Ruckel (IEH) calculations. The heme chromophores
are orientated such that transitions polarized in the heme
plane (x,y-polarized) can be readily distinguished from transitions
polarized perpendicular to the heme plane (z-polarized) . High spin
oxidized P450 exhibits two broad z-polarized bands, at 567 and
323 nm. IEH calculations suggest that these bands arise from
cysteine mercaptide sulfur-to-iron charge transfer transitions.
High spin reduced P450 has no z-polarized bands. IEH calculations
suggest that loss of these bands occurs because the cysteine sulfur
is protonated to a mercaptan. Low spin CO-P450 has an intense
x,y-polarized band at 363 nm. This transition, assigned as a mercaptide
sulfur-to-porphyrin charge transfer transition, has the
correct symmetry to mix with the Soret and may cause the
anomalous red shift of the Soret
EcoCyc: A comprehensive view of Escherichia coli biology
EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia coli biology. EcoCyc integrates information about the genome, genes and gene products; the metabolic network; and the regulatory network of E. coli. Recent EcoCyc developments include a new initiative to represent and curate all types of E. coli regulatory processes such as attenuation and regulation by small RNAs. EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a dataset of E. coli GO terms available through the GO Web site. The curation and visualization of electron transfer processes has been significantly improved. Other software and Web site enhancements include the addition of tracks to the EcoCyc genome browser, in particular a type of track designed for the display of ChIP-chip datasets, and the development of a comparative genome browser. A new Genome Omics Viewer enables users to paint omics datasets onto the full E. coli genome for analysis. A new advanced query page guides users in interactively constructing complex database queries against EcoCyc. A Macintosh version of EcoCyc is now available. A series of Webinars is available to instruct users in the use of EcoCyc
EcoCyc: a comprehensive database of Escherichia coli biology
EcoCyc (http://EcoCyc.org) is a comprehensive model organism database for Escherichia coli K-12 MG1655. From the scientific literature, EcoCyc captures the functions of individual E. coli gene products; their regulation at the transcriptional, post-transcriptional and protein level; and their organization into operons, complexes and pathways. EcoCyc users can search and browse the information in multiple ways. Recent improvements to the EcoCyc Web interface include combined gene/protein pages and a Regulation Summary Diagram displaying a graphical overview of all known regulatory inputs to gene expression and protein activity. The graphical representation of signal transduction pathways has been updated, and the cellular and regulatory overviews were enhanced with new functionality. A specialized undergraduate teaching resource using EcoCyc is being developed
An Improved, Bias-Reduced Probabilistic Functional Gene Network of Baker's Yeast, Saccharomyces cerevisiae
Background: Probabilistic functional gene networks are powerful theoretical frameworks for integrating heterogeneous functional genomics and proteomics data into objective models of cellular systems. Such networks provide syntheses of millions of discrete experimental observations, spanning DNA microarray experiments, physical protein interactions, genetic interactions, and comparative genomics; the resulting networks can then be easily applied to generate testable hypotheses regarding specific gene functions and associations. Methodology/Principal Findings: We report a significantly improved version (v. 2) of a probabilistic functional gene network [1] of the baker's yeast, Saccharomyces cerevisiae. We describe our optimization methods and illustrate their effects in three major areas: the reduction of functional bias in network training reference sets, the application of a probabilistic model for calculating confidences in pair-wise protein physical or genetic interactions, and the introduction of simple thresholds that eliminate many false positive mRNA co-expression relationships. Using the network, we predict and experimentally verify the function of the yeast RNA binding protein Puf6 in 60S ribosomal subunit biogenesis. Conclusions/Significance: YeastNet v. 2, constructed using these optimizations together with additional data, shows significant reduction in bias and improvements in precision and recall, in total covering 102,803 linkages among 5,483 yeast proteins (95% of the validated proteome). YeastNet is available from http://www.yeastnet.org.This work was supported by grants from the N.S.F. (IIS-0325116, EIA-0219061), N.I.H. (GM06779-01,GM076536-01), Welch (F-1515), and a Packard Fellowship (EMM). These agencies were not involved in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript.Cellular and Molecular Biolog
The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli
Background: Escherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses
A Genome-Wide Gene Function Prediction Resource for Drosophila melanogaster
Predicting gene functions by integrating large-scale biological data remains a challenge for systems biology. Here we present a resource for Drosophila melanogaster gene function predictions. We trained function-specific classifiers to optimize the influence of different biological datasets for each functional category. Our model predicted GO terms and KEGG pathway memberships for Drosophila melanogaster genes with high accuracy, as affirmed by cross-validation, supporting literature evidence, and large-scale RNAi screens. The resulting resource of prioritized associations between Drosophila genes and their potential functions offers a guide for experimental investigations
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