Biochemical Analysis of Psychrophilic Proteins from the Methanogen Methanococcoides Burtonii
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Biochemical Analysis of Psychrophilic Proteins
from the methanogen Methanococcoides burtonii
Erick Morales
Abstract
About 75% of our biosphere’s temperature is cold (≤ 5°C) and many microorganisms inhabit this type (psychrophilic) of environment, requiring the same building blocks for life as organisms inhabiting moderate (mesophilic) temperatures. Despite this, research on the biosynthetic pathways psychrophiles use is very limited. The enzymes found in these microbes are adapted with structural features that give them the ability to function up to 10 times higher catalytic efficiency than their mesophilic homologues. The overall goal of the proposed research is to use a biochemical and genetics approach to study the stability, structure, and function of psychrophilic proteins involved or believed to be involved in the nitrogen metabolism of Methanococcoides burtonii. The specific objectives are:
1. Investigate the structural and functional properties of M. burtonii’s nifH and nifD gene products using sequence analysis, UV-VIS spectroscopy, and two-hybrid studies;
2. Investigate the structure, stability, and kinetics of M. burtonii’s glutamate dehydrogenase through sequence analysis, site-directed mutagenesis, protein modeling, and kinetic assays.
Cold-enzyme study can increase knowledge in the field of protein folding and catalysis and the broader impacts of this project include (1) potential biotechnological applications of cold-enzymes (2) the involvement of high school students and science teacher(s) in hands-on research; and (3) the presentation of research results to a non-scientific audience in understandable terms.
Project Description
Research Objectives and Significance
About 75% of our biosphere’s temperature is permanently cold (≤ 5°C) and many microorganisms have adapted to this type (psychrophilic) of environment (1). Despite this, biochemical research on psychrophiles is limited, especially in archaea (2). One adaptation of these organisms is seen in their enzyme’s stability and structure, allowing for a higher catalytic efficiency than their mesophilic (moderate temperature) homologues (3). The recently sequenced genome of the archaeon Methanococcoides burtonii has revealed interesting features regarding its nitrogen metabolism, particularly the presence of nifH and nifD homologues but not other nif genes necessary for nitrogen fixation (4). Though unpublished results suggest a role in nitrogen fixation (4), other evidence suggests a role not involved in nitrogen fixation (5). Due to the uncertainty of the role of nifH and nifD in M. burtonii, it is of interest to determine their function. Furthermore, M. burtonii contains a gene for glutamate dehydrogenase, a well studied enzyme involved in nitrogen metabolism (6). In this project, it can serve as a model to study different adaptations to psychrophily in M. burtonii or corroborate those seen in psychrophilic bacteria (7). The overall goal of the proposed research is to use a biochemical and genetics approach to study the stability, structure, and function of psychrophilic proteins involved or believed to be involved in the nitrogen metabolism of M. burtonii. The specific objectives are: 1. Investigate the structural and functional properties of M. burtonii’s nifH and nifD gene products using sequence analysis, UV-VIS spectroscopy, and two-hybrid studies; 2. Investigate the structure, stability, and kinetics of M. burtonii’s glutamate dehydrogenase through sequence analysis, site-directed mutagenesis, protein modeling, and kinetic assays.
This research will contribute new biochemical data to the current limited knowledge on psychrophilic archaeal enzymes. The project can also increase knowledge in the field of protein folding and catalysis (8) and have several biotechnological applications such as in the industrial industry and food industry (3, 9-11).
Scientific Background
Psychrophilic enzymes. The study of psychrophilic enzymes is a relatively new, but growing field (12). These enzymes have adapted to the cold in a number of ways including maintaining their three-dimensional structure at cold temperatures and increasing activity up to ten fold more than mesophilic (moderate temperatures) enzymes (3). At the organism level, they offer one way for their adaptation to cold environments. At the biochemical level, they allow the slower metabolic processes to run with higher efficiency (8).
A few features that allow for a higher efficiency by psychrophilic enzymes have been found and corroborated mainly by studying bacterial psychrophilic enzymes (7). For instance, by studying the α-amylase of the bacterium Pseudoalteromonas haloplanktis, it has been found that cooperative unfolding is possible due to the simultaneous disruption of a limited number of interactions . Also, through folding studies using chimeric enzymes it was found that the active site of psychrophilic enzymes is the most flexible region where unfolding begins (8). It would be valuable to learn if this is true for archaeal psychrophilic enzymes, too.
The high flexibility at the active site in turn affects the binding affinity of substrates. Thus, psychrophilic enzymes generally have higher Km values than their mesophilic homologues. However, the catalytic rate (Kcat) is generally higher. Specific activity has been seen up to 10 times higher20. Taken together, the catalytic efficiencies (Kcat/Km) of psychrophilic enzymes are generally higher than their mesophilic homologues. This is one major way bacterial psychrophilic enzymes have adapted to cold temperatures (3).
Altogether, their study can help the understanding of cold-adaptation of organisms at the biochemical level, the understanding of enzyme folding and catalysis at a protein biochemistry level, and can provide applicable knowledge for biotechnological advancement.
Nitrogen Metabolism. Nitrogen is one of the most important elements required for life. Both proteins and nucleic acids, essential macromolecules, require nitrogen. Despite nitrogen’s importance for life, the study of nitrogen metabolism of particular organisms (e.g., psychrophiles) has been essentially absent for various reasons, including lack of technology (13). However, now that it is technologically possible to study the enzymes involved in nitrogen metabolism of psychrophiles, it is a good time to dedicate research in this area. As already mentioned, about 75% of our biosphere is cold and the psychrophilic organisms inhabiting these cold environments also need nitrogen. One particular area of low research is the nitrogen metabolism of archaea (13) so studying the proteins involved in the nitrogen metabolism of M. burtonii adds knowledge in two understudied areas with one effort.
Methanococcoides burtonii. M. burtonii was isolated from a sample of methane-saturated, saline water (1-2°C) of Ace Lake, Antarctica (14). It is a methylotrophic methanogen, requiring one-carbon substrates such as methanol and methylamines as carbon sources. It is flagellated, giving this psychrophile motility and the ability to go to a source of nutrients when needed. Its temperature growth range is from -2°C to 28°C with an optimum at 23°C (8). This is important because enzymes might not be significantly affected by temperatures of up to 25°C and so there is a god chance for E. coli cells to be grown and expression to be possible at not so cold temperatures. Other organsism with lower growth ranges may have enzymes that denature at even 15°C. This methanogen has been studied to a some extent. However, biochemical studies on proteins of this or any other archaeal prychrophile is limited to only two as of the year 2000, both enzymes involved in DNA replication (15-16). Thus, to have a more clear understanding of psychrophily and the proteins and mechanisms involved more research must be dedicated to analysis of the proteins of psychrophilic archaea and M. burtonii offers a good model system.
Glutamate dehydrogenase (GDH). There are two types of GDHs currently known. One is a tetramer requiring NAD+ as the cofactor. The other is a hexamer, requiring only NAD+, or only NADP+, or either. Since GDH is important in nitrogen metabolism, is found in all domains of life, and is a well studied enzyme, its study from a psychrophilic archaeon is valuable, especially since there is limited information on this archaeal form. The wealth of information and the data from a known psychrophilic GDH from a bacterium (6) can serve as models for studying M. burtonii’s GDH. Just as importantly, comparative analysis of information on M. burtonii’s GDH and the GDHs from the rest of the domains can help study the enzymatic biochemical adaptation of cold temperatures (6).
The study of the bacterial GDH has revealed important findings. One thing is that unlike most psychrophilic enzymes, the GDH from the bacterium psychrobacter sp. TAD1 does not conform to the generally features of psychrophilic enzymes. The psychrobacter sp. TAD1 does not have high catalytic efficiency at low or moderate temperatures (6).
Another interesting aspect of pscychrobacter sp. TAD1 GDH is its kinetics. The GDH of pscyrhobacter sp. TAD1 shows positive cooperativity with α-ketoglutarate and NADPH (the reverse reaction), which is not typically seen in prokaryotic GDHs (6). It will be interesting to determine if this is also true for M. burtonii’s GDH, which if true, will suggest that this type of activity may be important in psychrophilic GDHs. The results of the study of M. burtonii’s GDH may offer the first comparison of an archaeal psychrophilic GDH to bacterial psychrophilic GDH and mesophilic GDHs.
nifH and nifD homologues. These term “nif’ is used in the naming of these genes because of their nitrogen fixation role. The proteins nifH and nifD are normally part of the nitrogenase complex that essentially consists of two enzymes responsible for nitrogen fixation. nifH codes for one enzyme, called nitrogenase reductase. It is almost always a homodimer that contains a single [4Fe-4S] cluster that connects the two subunits. Its function is to hydrolyze ATP and transfer, via the Fe-S cluster, one electron donated by ferredoxin to the other enzyme called nitrogenase. Nitrogenase, which actually reduces N2 to NH3, is a heterotetramer composed of two identical α subunits and two identical β subunits coded respectively by nifD and nifK. nifD codes for the α subunits and contains a molybdenum-iron cofactor, which accepts the electrons from the Fe-S cluster of nitrogenase reductase. nifK codes for the two β subunits (17). In M. burtonii, nifK is not present and this is one of the reasons it is believed that M. burtonii does not fix nitrogen. One particular archaeon, Methanosarcina barkeri, has been found to contain a homotetrameric nitrogenase reductase instead of the common homodimeric reductase (18). It would be interesting to determine if this is the same or different with the nitrogen reductase, nifH, of M. burtonii.
The main purpose of studying the products of the nifH and nifD homologues is to determine their function in M.burtonii and determine whether or not they are involved in nitrogen fixation or not.
Research Plan
Objective 1: To determine structural and functional properties of M. burtonii’s nifH and nifD gene products using sequence analysis, UV-VIS spectroscopy, and two-hybrid studies.
Sequence Analysis Since nifH contains an Fe-S cluster and an ATP binding site, two sequence analyses can be carried out. One, a multiple sequence alignment of M. burtonii’s nifH against other nifH genes can be done to determine whether the two conserved cysteine residues are present as they are in all nifH proteins (5). The second multiple sequence alignment can work to determine if nifH contains a binding site for ATP. Since there is an ATPase motif, this can be done.
Expression To express nifH and nifD, the first step will be to genetically engineer the genes into an expression system. There are a couple of ways to do this. One common way is to genetically engineer the genes into the pet15b (His6-tag) and pET41a (no His6-tag) expression vectors, which will then be transformed into E. coli expression lines. Cells will be grown at 15°C and after cell breakage, protein solubility will be analyzed. If there is no induction or very low protein solubility, then an alternative expression system will be used. This is the use of Clone Q vector and the psychrophilic host Pseudoalteromonas haloplanktis TAC 125 to express genes from psychrophiles (19). Procedures will be changed as needed to get soluble protein.
During and after cell breakage, procedures will be carried out under anaerobic conditions.
Protein Analysis. Soluble proteins will be analyzed by SDS-PAGE to determine correct molecular weight. If so, further analysis by UV-VIS spectroscopy of the reduced and oxidized forms will confirm the presence of the iron-sulfur clusters. To determine if these proteins accept electrons, reducing agents such as ferredoxin or dithionite may be used to analyze this. Furthermore, a bacterial two-hybrid study and the β-galactosidase assay will be used as Staples et al did when the nifH and nifD homologues of Methanocaldococcus jannaschii were analyzed for protein-protein interactions (5).
Predicted Outcomes
The study of the nifH and nifD homologues in M. jannaschii revealed that they did interacted with each other. Considering the similarities between M. jannaschii and M. burtonii in being methanogens and not having other important nitrogen-fixation genes, it is predicted that M. burtonii’s nifH and nifD will interact with each other, too. This will suggest a role outside of nitrogen fixation, though perhaps one similar to it (e.g., electron transferring).
Objective 2: Investigate the structure, stability, and kinetics of M. burtonii’s glutamate dehydrogenase through sequence analysis, site-directed mutagenesis, protein modeling and kinetic assays.
Since mesophilic glutamate dehydrogenase is a well-studied enzyme and a bacterial psychrophilic glutamate dehydrogenase has been studied (6 ), studying M. burtonii’s glutamate dehydrognease can serve for comparison reasons. The bacterial psychrophilic glutamate dehydrogenase is especially useful because some procedures can be modeled after it.
During and after cell breakage, procedures will be carried out under anaerobic conditions.
Expression. Will be followed in similar fashion as above for the nifH and nifD gene expression.
Activity and Stability studies. After producing soluble glutamate dehydrogenase, its activity will be analyzed with the glutamate dehydrogenase assay (6). Its optimum pH and temperature will be determined.
Protein modeling will be performed using protein modeling software and using crystal structures from mesophilic glutamate dehydrogenases. There is currently no crystal structure for a psychrophilic GDH.
Kinetic Studies. Will be carried after the glutamate dehydrogenase assay is well developed. A series of increasing concentrations of substrate (glutamate), keeping the concentration Mg2+ and ATP constant will be used.
Site-directed mutagenesis. After sequence alignment against the bacterial psychrophilic GDH (Psychrobacter sp. TAD1) and mesophilic GDHs, conserved amino acid residues in the psychrophilic enzymes will be identified. Thereafter, an alanine screen and conservative mutations will be carried out.
Product Inhibition Studies. Time permitted, product inhibition studies will also be carried out.
Predicted Outcomes. It is predicted that M. burtonii’s glutamate dehydrogenase will have more similar biochemical properties to the psychrophilic bacterial glutamate dehydrogenase. However, it has been observed that enzymes, though psychrophilic but from different organisms, do not always have similar biochemical properties (6). This is why it is useful to carry out these experiments.
Broader Impacts
High school student participation. High school students will directly participate in the research. Specifically, they will first start off by learning safety and basic laboratory techniques (pipette use, etc.). They will then, based on their preference, perform (1) genetic engineering on site-directed mutant genes or (2) grow cells, induce, and purify GDH variants in order to carry out GDH assays. Based on time and motivation, they may also perform kinetic studies on GDH.
Biotechnological applications. The study of psychrophilic enzymes has already a number of biotechnological applications. For example, in the industrial “peeling” of leather, psychrophilic proteases may be used instead of mesophilic proteases so that regular tap water can be used instead of heating up to 37°C (3). In the food industry, there already is a patent for the use of psychrophilic β-galactosidase so that it could be used to remove lactate in milk (3). The proposed research will contribute to the understanding of psychrophilic enzymes and increase knowledge for other biotechnological applications.
Qualifications. Performed research in a biochemistry lab for almost two years at California State University, Fullerton. Research experience includes sequence analysis, genetic engineering, purification of His6-tagged proteins, protein analysis (Bradford assay, SDS-PAGE), enzymology (use of an isothermal titration calorimeter to determine kinetic constants, running assays), and working in anaerobic conditions. Since one of my career goals is to become a high school science teacher (biology, chemistry, and physics), the research will enable me to both continue my interest in research and encourage students to enter a science field.
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