Created by James Beaton
Alanine racemase, (PDB ID: 2SFP), is a 88033.45 Da protein with an isoelectric point (pI) of 6.62, and is found in Bacillus stearothermophilus. The function of alanine racemase is the catalysis of the racemization of L-alanine to D-alanine, as well as the back reaction from D-alanine to L-alanine (1). Therefore, alanine racemase is an isomerase and an epimerase in addition to simply be a racemase. The classification of isomerase is due to the fact that a different stereoisomer of alanine is obtained after its interaction with alanine racemase. The classification of epimerase is because alanine racemase catalyzes the rearrangement of the hydrogen group and the hydroxyl group of the last asymmetric carbon in the exchange of one conformation of alanine for the other (2). This catalasys is crucial to the formation of the peptidoglycan layer of cell walls, which require D-alanine, the less common natural enantiomer of alanine. Specifically, D-alanine dipeptide is added to UDP-N-acetylmeramyl-L-Ala-D-Glu-meso-diaminopimelatte during the biosynthesis of the peptidoglycan layer of the cell wall (1). The peptidoglycan layer of bacterial plasma membranes is composed of both sugars and amino acids, one of which is D-alanine. The peptidoglycan layer functions in two ways. The first is to provide structural support for the plasma membrane, while the second is to help the bacterial cell cope with the osmotic pressure generated by the cytoplasm.
The function of alanine racemase in the formation of bacterial cell walls makes it a potential target for antibiotics, which would function as inhibitors in order to prevent the production of bacterial peptidoglycan layers, thus preventing the formation of complete bacterial walls, which prevents bacterial cell replication. However, because alanine racemase is a PLP dependant enzyme, none of the methods of inhibition that would be employed by potential pharmaceutical developers are specific enough to inhibit alanine racemase alone. This is because a variety of enzymes bind PLP as a ligand, meaning that any drug that has been developed as of now would target any enzyme that binds PLP in its active site, rather than being specific enough to impede the function of only alanine racemase (1).
The structure of alanine racemase was determined through X-ray crystallography at a resolution of 1.90 Å. The enzyme was found to be a homodimer in solution, and consists of two strands,an α-strand and a β-strand, each with a mass of 43,300 Da. Both the α-strand and β-strand are identical. Each of the strands contains an α/β domain at the N-terminus and a β-strand domain at the C-terminus. In total, alainine racemase contains 388 residues (1). Each monomer of alanine racemase contains two domains. The first domain consists of amino acids 1-240 and is an eight-stranded α/β barrel at the N-terminus. Amino acids 241-388 comprise the C-terminal domain, which is almost entirely made up of β-strand segments (1).
At the level of tertiary structure, the mouth of the α/β strand of one monomer is aligned with the β-strand portion of the other monomer. This is the area where the coenzyme PLP binds (1). In total, alanine racemase is 32% helical in secondary structure, containing 19 helices totaling 128 residues. Additionally, 26% of the secondary structure is made up of beta sheets: a total of 23 sheets containing 103 residues. During the determination of the structure of alanine racemase through X-ray crystallography, PLP was bound in the active site as an inhibitor (1).
The active site of alanine racemase, which binds the coenzyme PLP, contains residues from both the α-strand and the β-strand (1). The binding site for PLP is largely located in the α/β barrel at the N-terminus, and is linked to Lys-39 through an aldimine linkage (1). Therefore, it is shown that the structure of alanine racemase directly influences its function, as the binding site for the coenzyme PLP is located within the folds of the secondary and tertiary structural elements of the enzyme as a whole. However, the PLP dependence of alanine racemase means that there is not a specific substrate or inhibitor that binds to the enzyme, meaning that the details of the mechanisms of the active site are currently unknown. That being said, it is known that the active sites of both monomers of alanine racemase have PLP molecules bound to Lys-39 as a protinated Schiff base through an aldimine linkage. This action works in combination with the catalytic base action of Tyr-265 to remove an α-hydrogen from the alanine molecule that is being converted from one configuration to another (8).
This catalysis between the amino acids Lys-39 and Tyr-265 occurs between two separate amino acids so that once an alanine molecule is accepted; it is transferred to the other active site so that it can be reprotinated from the opposite side, thus changing the conformation of alanine. When L-alanine is the starting reactant and D-alanine is to be the product, the L-alanine binds first to the Tyr-265, and the alanine molecule is transferred to Lys-39 after deprotination. Conversely, when D-alanine is the starting reactant and L-alanine is to be the product, the alanine molecule binds first to Lys-39, and is transferred to Tyr-265 following deprotination of the α-carbon (8). It is also known that the two active sites are catalytically equivalent, although it is possible that one is completely inactive (1). It is also important to note that the residue Lys-129 is carboxylated, and contributes to the active site of alanine racemase (1).
In addition to the active site residue, Lys-39, alanine racemase containsother amino acids that have properties specific to the enzyme. The first is Lys-129. Lys-129 is capable of forming a carbamate on its side chain amino group. In addition, the formation of the carbamate on the side chain amino group of Lys-129 is stabilized by another residue: Arg-136. Finally, Tyr-265 is also involved in the catalysis of the exchange of L-alanine for D-alanine by acting as a proton acceptor for L-alanine (1).
Under a specific set of conditions including acidic conditions and incubation with L-alanine, alanine racemase can also catalyze a side reaction: transamination. Additionally, it was found that a mutant form of the enzyme exists, in which Lys-39 has been substituted for Ala-39. This mutated form of the enzyme is incapable of catalyzing either the normal conversion of L-alanine to D-alanine or the transamination side reaction under normal biological conditions. However, the addition of methylamine allowed both the normal reaction and the side reaction to proceed, which showed that the active site for the conversion of L-alanine to D-alanine, Lys39, is also the active site of the transamination side reaction (8).
Alanine racemase is a PLP-dependant enzyme, meaning that pyridoxal-phosphate, also known as PLP, acts as a coenzyme in the reaction catalyzed by alanine racemase. In PLP-dependant enzymes, the binding of PLP as a cofactor changes the conformation of the enzyme into the catalytically active form. In the case of alanine racemase, the alcohol group ofPLP is hydrogen-bound to the side chain amine group of Lys-39A, the two free phosphodiester oxygens of PLP are hydrogen-bound to the side chain alcohol groups of both Tyr-43A and Tyr-354A while the oxygen hydrogen-bound toTyr-43A also shares a hydrogen bond with the side chain amine group of Ile-222A , two hydrogen bonds are formed between the phosphodiester alcohol group of PLP and the side chain alcohol and amine groups of Ser-204A, and finally, the aromatic nitrogen of PLP is hydrogen bound to the side chain amine group of Arg-219A (1). In addition, an aldimine linkage is formed between PLP and Lys-39, which is part of the C-terminal α/β barrel (1).
Propanoic acid, or PPI, is alsobound to alanine racemase as a ligand. In this interaction, one of the carboxyl oxygen molecules shares two hydrogen bonds, one with the side chain alcohol of Tyr-265B and the other with the side chain amine group of Arg-136A, and the other carboxyl oxygen molecule is hydrogen bound to the side chain amine group of Met-312B (1).
The primary schematic for the conversion of L-alanine to D-alanine catalyzed by alanine racemase involves the displacement of Lys-39 during a transaldimination. This transaldimination results in a geminal diamine intermediate, which is converted to an external aldimine intermediate, followed by a quinonoid carbanion intermediate. The α-carbanion is then protinated from the back side, resulting in the exchange of one enantionmer of alanine for the other. Finally, alanine in its new conformation dissociates when a transaldimination occurs at Lys-39, which displaces the alanine molecule (1).
Analysis of alanine racemase (PDB ID 2sfp), found in Bacillus stearothermophilus, using PSI-BLAST returned several resulting proteins that are very similar in primary structure to alanine racemase (PDB ID: 2sfp). The first comparison protein that was found was alanine racemase (PDB ID: 3HA1), which is found in the organism Bacillus anthracis, and had an E-value of 4?10-154, showing that the number of differences in the amino acid sequence between alanine racemase (PDB ID: 2sfp) and alanine racemase (PDB ID: 3HA1) is very small (4). Comparison of the tertiary structures of alanine racemase (PDB ID: 2sfp) and alanine racemase (PDB ID: 3HA1) through the use of a Dali Server search returned a Z-score of 53.5, showing that in addition to similar primary structures, the two proteins also had very similar folding patterns and tertiary structures (5).
Overall, the amino acid sequence of alanine racemase (PDB ID 3HA1) is 56.44% identical and 71.13% similar to the amino acid sequence of alanine racemase (PDB ID 2sfp). Additionally, the tertiary structure of alanine racemase (PDB ID 3HA1) is 98% similar to that of alanine racemase (PDB ID 2sfp), and its secondary structure is comprised of 30% helices and 26% beta sheets (1).
Unlike alanine racemase (PDB ID 2sfp), alanine racemase (PDB ID 3HA1) is not a PLP-dependent enzyme. In fact, PLP is not even one of its ligands. Instead, alanine racemase (PDB ID 3HA1) bindstwo ligands: acetate ion (ACT) and chloride ion (CL) (1). The chloride ion acts to change the conformation of alanine racemase (PDB ID 3HA1) in order to allow it to catalyze the conversion of L-alanine to D-alanine, which is the function of PLP in alanine racemase (2sfp) (7). Additionally, alanine racemase (PDB ID 3HA1) is longer than alanine racemase (PDB ID 2sfp) by 9 amino acids, and weighs 90144.39 Da, which is slightly greater than alanine racemase (PDB ID 2sfp) (1). Like alanine racemase (PDB ID 2sfp), alanine racemase (PDB ID 3HA1) is targeted by drugs in order to prevent cell wall development of the species that it is found in: Bacillus anthracis, which is the species responsible for causing anthrax. Like alanine racemase (PDB ID 2sfp), alanine racemase (PDB ID 3HA1) is a dimer in solution (7).
The second comparison protein was alanine racemase (PDB ID 3E5P), found in Enterococcus faecalis, and was found to be sequentially and structurally similar to alanine racemase (PDB ID 2sfp). Examination using PSI-BLAST returned an E-value of 3?10-117, showing that the primary structure was very similar to that of alanine racemase (PDB ID 2sfp), and examination using a Dali Server search returned a Z-score of 49.2 showing that the two proteins had similar folding patterns and tertiary structures (4,5).
Direct comparison of the amino acid sequences shows that alanine racemase (PDB ID 3E5P) was 47.68% identical and 61.60% similar to alanine racemase (PDB ID 2sfp). Additionally, the tertiary structures were 99% similar. However, alanine racemase (PDB ID 3E5P) contains three chains, A, B, and C, while alanine racemase (PDB ID 2sfp) contained only A and B chains (1). Finally, the secondary structure of alanine racemase (PDB ID 3E5P) is 30% helical and comprised of 23% beta sheets, very close to the respective 32% and 26% values of alanine racemase (PDB ID 2sfp) (1).
Similar to alanine racemase (PDB ID 2sfp), alanine racemase (PDB ID 3E5P) is a PLP dependent enzyme (6). Additionally, it is about 1/3 heavier than alanine racemase (PDB ID 2sfp) with a weight of 124722.06 Da, which is most likely due to it having three strands that are each similar to the strands of alanine racemase (PDB ID 2sfp), which only has two strands (1). In addition to the ligands that alanine racemase (PDB ID 2sfp) binds, pyridoxal-5’-phosphate (PLP) and propanoic acid (PPI), alanine racemase (PDB ID 3E5P) binds two additional ligands: nonethylene glycol (2PE) and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (EPE) (1). Similar to alanine racemase (PDB ID 2sfp), one of the main active sites is a tyrosine, and is located at 44, which is very close to the active site of alanine racemase (PDB ID 2sfp), which is located at Tyr-43(6). Finally, alanine racemase (PDB ID 3E5P) is a target for potential antibiotics to those that are attempting to be developed to inhibit alanine racemase (PDB ID 2sfp) as antimicrobial agents that prevent the formation of bacterial cell walls (3).
The final protein used for comparison was alanine racemase (PDB ID 3OO2), which, when compared to alanine racemase (PDB ID 2sfp) using a PSI-BLAST search returned an E value of 1?10-94, and returned a Z-score of 48.0 when examined using a Dali Server search (4,5). The amino acid sequence of alanine racemase (PDB ID 3OO2) is 42.27% identical and 59.79% similar to that of alanine racemase (PDB ID 2sfp), while the Folding patterns and tertiary structure are 98% similar (1).
Unlike alanine racemase (PDB ID 2sfp), alanine racemase (PDB ID 3OO2) is not a PLP dependant enzyme. Instead, it binds three ligands, all of which are different that those of alanine racemase (PDB ID 2sfp). They are beta-mercaptoethanol (BME), sodium ion (NA), and phosphate ion (PO4). Alanine racemase (PDB ID 3OO2) differs in length from alanine racemase (PDB ID 2sfp) by just three residues, and weighs 86766.11 Da, which is very close to the weight of alanine racemase (PDB ID 2sfp). Additionally, studies are still being conducted on alanine racemase (PDB ID 3OO2) in order to determine information about it such as its active site and how the ligands affect the catalysis of the conversion of L-alanine to D-alanine (1).
In all, alanine racemase is a very interesting protein, which serves an important function in the replication of many different kinds of cells. This means that one day it may be the target of widely used drugs, which may help save many lives.