Overview
MethanE.COLIc project is designed to solve one of the problems of Turkey on worker security in mines, by constructing the natural parts of organisms. This project focuses on sensing the ambient methane gas and converting to one of the bio-fuel sources, methanol. Methanol is entrapped by product of one of the devices of project ; then to elute methanol, cells are dead by kill switch device of project.
The main reason for us to choose such a project is that in Turkey- and also in many countries- each year, huge numbers of workers in mines lost their lifes due to deficiencies and conditions in their working areas (mines). The explosions due to revealed methane gas which is named as Grizu explosions leads to subsidence of mine
Subtitles
1. Methane sensing
- Introduction
In our methane.colic project, the first critical point is to sense the ambient methane gas. While in literature search, we have focused on the methane and DNA or methane and regulator protein interaction which binds to DNA after binding to methane. In structure methane consists of one carbon atom and four hydrogen atom which means it is smaller in size and weight. Therefore it is hard to find any study based on methane interaction with DNA or protein to regulate transcription. This made us search for carbon hydrogen bond interaction with any oligo or protein to trigger transcription of any metabolite product. So that we also searched the literature for any organism which utilizes any alkane chain and alkane which regulates the transcription of organismal metabolites by interaction with DNA or protein. Since bacteria are environmental adapting organisms, it is possible to find any alkane to regulate the transcription of degradation metabolism. Then we have found the organism, Pseudomonas oleovorans to analyze the alkane degradation of organism for its carbon source.
- Background
Many microorganisms live in the environments where the conditions changing frequently and so the evolution is inevitable for mechanisms to withstand unfavorable situations. Therefore microorganisms can use their specific and sensitive mechanisms for sensing the required nutrients for them or any pollution to affect their sustainity. Otherwise they can expose to mutations which change the gene expression and gain new functionalities. In case they have ability to survive in such conditions.
While we were scanning the literature based on methane and alkane degrading organisms we have found some organisms that sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.
We analyzed the soil bacteria, Pseudomonas oleovorans. This strain can assimilate the alkane for its carbon source and one of the microbial whole cell –biosensor. They have a gene cluster which codes for degradative pathway and includes the activator which interacts especially with linear alkanes. This activator protein, AlkS, in the presence of alkanes, induces the transcription from PalkB promoter which initiates the expression of genes code for assimilation of alkanes. We have analyzed the related articles[REFERENCE] in which the promoter region is studied and showed that it is expressed in E.coli correctly because of the corresponding RNA polymerase binding regions.
- Modelling
2. Conversion
Molecular biology and regulation of methane monooxygenase
J. Colin Murrell · Bettina Gilbert · Ian R. McDonald, 2000, Molecular biology and regulation of methane monooxygenase
Methanotrophs - methane oxidizing bacteria- are ubiquitous in the environment and play an important role in mitigating global warming because they are a unique group of gram-negative bacteria that grow aerobically on methane as sole source of carbon and energy (Hanson and Hanson 1996). They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. The first step in the oxidation of methane is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO). sMMO components have been expressed in heterologous and homologous hosts. The pMMO has been purified and biochemically studied in some detail and the genes encoding the pMMO have been sequenced. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes.
--Soluble Methane Monooxygenase
In contrast to pMMO, sMMO has extremely broad substrate specificity and can oxidise a wide range of non-growth substrates such as alkanes, alkenes and aromatic compounds thus making it the more attractive enzyme for co-oxidation reactions and bioremediation processes (Sullivan et al. 1998). sMMO is expressed only under conditions in which the copperto-biomass ratio is low, i.e. under “low-copper” growth conditions, when copper ions are omitted from the trace elements solution of a standard mineral salts medium or cells are grown in a fermentor to high cell densities (OD>6.0; see below). There is also some evidence that copper ions inhibit the activity of sMMO (Jahng and Wood 1996). Like many other multi-component oxygenase systems, sMMO contains a component of approximately 16 kDa, Protein B, which serves an “effector” or regulatory role. The activity of Protein B may be regulated by proteolysis at its amino terminus (Lloyd et al. 1997). At low concentrations, Protein B converts the hydroxylase from an oxidase to a hydroxylase and stabilises intermediates necessary for oxygen activation. Saturating amounts of Protein B dramatically increase the rates of formation of intermediates and accelerate catalysis of methane to methanol by sMMO (Lee and Lipscomb 1999). The structure of Protein B from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) has recently been obtained by NMR spectroscopy (Walters et al. 1999). The most extensively characterised sMMO enzymes are those from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b (reviewed in Lipscomb 1994; Deeth and Dalton 1998). The sMMO is a non-haem iron-containing enzyme complex consisting of three components. The hydroxylase consists of three subunits of 60, 45 and 20 kDa arranged in an α2 β2 γ2 configuration. Like many other multi-component oxygenase systems, sMMO contains a component of approximately 16 kDa, Protein B, which serves an “effector” or regulatory role. At
low concentrations, Protein B converts the hydroxylase from an oxidase to a hydroxylase and stabilises intermediates necessary for oxygen activation. Saturating amounts of Protein B dramatically increase the rates of formation of intermediates and accelerate catalysis of methane to methanol by sMMO (Lee and Lipscomb 1999). sMMO genes are clustered on the chromosome of Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. mmoX, mmoY and mmoZ encode the α-, β- and γ-subunits respectively of the hydroxylase. mmoB and mmoC code for Protein B and the reductase component. Interestingly, mmoB lies between mmoY and mmoZ; an ORF of unknown function, designated orfY, with a coding capacity of 12 kDa, lies between mmoZ and mmoC in all genes clusters analysed to date (McDonald et al. 1997).
-- Particulate methane monooxygenase
The pMMO consists of three subunits of approximately 45, 27 and 23 kDa (Zahn and DiSpirito 1996; Nguyen et al. 1998) in a stoichiometry of 1:1:1. The 45 and 27 kDa polypeptides probably contain the active site because they are specifically labelled by [14 C]-acetylene, a suicide substrate for MMO (Prior and Dalton 1985; Zahn and DiSpirito 1996). The active enzyme contains 2 iron and approximately 15 copper atoms per mol.
Effect of n-Alcohols on the Structure and Stability of the Drosophila Odorant Binding Protein LUSH
Brigid K. Bucci, Schoen W. Kruse, Anna B. Thode, Sylvia M. Alvarado, and David N. M. Jones
LUSH is an alcohol-sensitive odorant binding protein expressed in the olfactory organs of Drosophila melanogaster, and it is used as a model system to investigate the biophysical nature of
alcohol-protein interactions at alcohol concentrations that produce intoxication in humans. In this study, by using NMR spectroscopy, they have identified the regions of LUSH that show increased conformational stability on binding alcohols. These residues primarily line the alcohol-binding pocket. A direct measure of the degree of stability that alcohol imparts on LUSH has been provided. LUSH was originally identified as responsible for mediating an avoidance response to short-chain n-alcohols.
The general structure of odorant binding proteins consists of six α-helices surrounding a hydrophobic ligand- binding pocket which differs in size and shape between each protein. All these odorant binding proteins have a set of six cysteines that form three conserved disulfide bonds. In the study, by observing the X-ray crystal structures of LUSH-alcohol complexes, it was found that alcohol binds to a single site in the protein formed by a network of concerted hydrogen-binding residues located at one end of hyrophobic pocket. This binding site has some sequence and/or structural similarities to regions of several ligand gated ion channels (LGICs) that have previously been implicated in inferring sensitivity to alcohol. It is hypothesized in the study that the alcohol-binding site in LUSH may represent a more general structural motif for functionally relevant alcohol-binding sites in proteins. The characterization of the effects of n-alcohols on the structure and stability of LUSH is presented. Also, in the absence of ligand, LUSH exists in vitro in a partially unstructured state and binding of alcohols shifts the solution conformation to a more compact folded state which is accompanied by an increase in the overall protein stability. Those regions of the protein that show the largest changes in local dynamics on binding alcohol have been identified and it have been shown that these are predominantly associated with the residues that line the alcohol-binding pocket. The results provide a quantitative measure of the ability of short-chain alcohols to stabilize protein structure at physiological relevant concentrations.
The role of multiple hydrogen bonding groups in specific alcohol binding sites in proteins: Insights from structural studies of LUSH
In this study, the effects of specific amino acid substitutions on alcohol binding have been designed and tested according to information from previously solved crystal structures of Drosophila melanogaster protein LUSH in complexes with short chain alcohols.
