A major focus of our research group is to understand the bioorganic chemistry and biochemistry of the amino acid methionine and its roles in proteins, cellular pathways and its function as S-adenosyl-L-methionine (AdoMet).
Methionine analogs
Methionine is one of the standard twenty amino acids found routinely in proteins. We have synthesized a number of unnatural analogs of methionine, two of which are shown. Difluoromethionine (DFM) and trifluoromethionine (TFM) are stable analogs of methionine and were synthesized to study the effect of fluorination on the electronic properties of the sulfur atom in this amino acid. For example, a number of metal centers in metalloenzymes have the sulfur of a methionine residue as a ligand. We are interested in the effects that fluorination would have on these metal centers. Additionally, the fluorine atoms in these molecules serve as NMR sensitive nuclei useful in the application of 19F NMR biophysical techniques to study protein structure and function. Other types of methionine analogs are being synthesized and studied for their biochemical, biophysical and medicinal chemistry properties as well as in the preparation of proteins with novel properties.
Effects of fluorination on the calculated electronic properties of the thiomethyl group (from left to right): CH3SCH3, CHF2SCH3 and CF3SCH3)
Other methionine analogs that can be further modified by a variety of chemical methods (shown below) are being studied for their application in protein engineering.
Methionine in proteins
Incorporation of methionine into proteins occurs through a number of enzyme steps as outlined in the figure. We are exploring several of these enzymes. For example, we have studied the enzyme methionyl-tRNA synthetase (MetRS) with methionine analogs, and we have incorporated unnatural methionine analogs into several proteins. Other enzymes that we are exploring include methionine aminopeptidase (MetAP) which removes N-terminal methionine residues from proteins and inhibition of which can lead to new antibacterial and anticancer agents. The enzyme methionine sulfoxide reductase (MSR) is also being studied to explore the chemical mechanism of this enzyme, which reduces oxidized methionine residues in proteins from the sulfoxide to the sulfide form. This enzyme is also of interest since some pathogenic organisms use their own MSR to protect themselves from the human immune system and also to control their pathogenicity.
Cellular pathways
There are various cellular pathways that involve the amino acid methionine. One such pathway is the conversion of methionine into alpha-ketobutyrate, ammonia and methanethiol in certain pathogenic organisms such as Entamoeba histolytica (cyst form shown) and Trichomonas vaginalis. The enzyme, methionine gamma lyase (MGL), is a pyridoxal phosphate utilizing enzyme. We are studying the chemical mechanism of this enzyme to further understand the fundamentals of enzyme catalysis as well as to learn how we can use this knowledge to uncover new approaches to treat these pathogenic microorganisms.
Photo credit: Centers for Disease Control and Prevention, USA
AdoMet
Methionine, unlike most other amino acids, also has an active role to play in metabolism and cellular physiology. It undertakes these roles in the form of S-adenosyl-L-methionine (AdoMet). AdoMet is formed by the action of the enzyme ATP: L-methionine S-adenosyltransferase, which catalyzes the reaction of methionine with ATP to produce AdoMet, Pi and PPi as products. AdoMet can serve as a source for active methyl groups that can be transferred to various acceptor molecules such as DNA, RNA, protein, phospholipid and small molecules. We are investigating one of these reactions which involves the study of an enzyme that leads to antibiotic resistance.
AdoMet (left) and Thiostrepton (right)
The thiopeptide antibiotic thiostrepton is biosynthesized by a strain of Streptomyces. Thiostrepton is very active against certain microorganisms and acts by binding tightly to bacterial ribosomes, halting protein biosynthesis. In order to protect itself against the action of the antibiotic that it makes, the organism uses a methyltransferase, thiostrepton resistance methyltransferase, to transfer a methyl group from AdoMet to a specific adenosine in the Streptomyces 23S ribosomal RNA. This methylation sterically blocks binding of thiostrepton to the ribosome, which protects the microorganism from inhibition of its own protein biosynthesis machinery.
In collaboration with Professor Graeme Conn (Emory University, USA) and his research group, the three-dimensional structure of this methyltransferase has been determined. We are currently studying the chemical mechanism of this enzyme and attempting to elucidate the functional roles of various amino acids in the active site of this enzyme. Additional studies on the chemical modification of thiostrepton to produce new analogs of this complex antibiotic are also underway. It is hoped that we can use our findings not only to better understand protein and antibiotic interactions with bacterial ribosomes but also to possibly discover improved antibacterial agents.
Thiostrepton (spacefill) bound to the large ribosomal subunit of D. radiodurans (PDB 3CF5).