Summary: Alanine racemase, N-terminal domain
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Alanine racemase Edit Wikipedia article
The 1.45Å a crystal structure of alanine racemase from Pseudomonas aeruginosa, PDB 1rcq
|PDB structures||RCSB PDB PDBe PDBsum|
|Gene Ontology||AmiGO / EGO|
the 1.45Å a crystal structure of alanine racemase from a pathogenic bacterium, pseudomonas aeruginosa, contains both internal and external aldimine forms
- L-alanine D-alanine
This enzyme belongs to the family of isomerases, specifically those racemases and epimerases acting on amino acids and derivatives. The systematic name of this enzyme class is alanine racemase. This enzyme is also called L-alanine racemase. This enzyme participates in alanine and aspartate metabolism and D-alanine metabolism. It employs one cofactor, pyridoxal phosphate. At least two compounds, 3-Fluoro-D-alanine and D-Cycloserine are known to inhibit this enzyme.
The D-alanine produced by alanine racemase is used for peptidoglycan biosynthesis. Peptidoglycan is found in the cell walls of all bacteria, including many which are harmful to humans. The enzyme is absent in higher eukaryotes but found everywhere in prokaryotes, making alanine racemase a great target for antimicrobial drug development. Alanine racemase can be found in some invertebrates.
Bacteria can have one (alr gene) or two alanine racemase genes. Bacterial species with two genes for alanine racemase have one that is continually expressed and one that is inducible, which makes it difficult to target both genes for drug studies. However, knockout studies have shown that without the alr gene being expressed, the bacteria would need an external source of D-alanine in order to survive. Therefore, the alr gene is a feasible target for antimicrobial drugs.
To catalyze the interconversion of D and L alanine, Alanine racemase must position residues capable of exchanging protons on either side of the alpha carbon of alanine. Structural studies of enzyme-inhibitor complexes suggest that Tyrosine 265 and Lysine 39 are these residues. The alpha-proton of the L-enantiomer is oriented toward Tyr265 and the alpha proton of the D-enantiomer is oriented toward Lys39 (Figure 1).
The distance between the enzyme residues and the enantiomers is 3.5A and 3.6A respectively. Structural studies of enzyme complexes with a synthetic L-alanine analog, a tight binding inhibitor  and propionate  further validate that Tyr265 and Lys39 are catalytic bases for the reaction,.
The PLP-L-Ala and PLP-D-Ala complexes are almost superimposability. The regions that do not overlap are the arms connected the pyridine ring of PLP and the alpha carbon of alanine. An interaction between both the phosphate oxygen and pyridine nitrogen atoms to the 5’phosphopyridoxyl region of PLP-Ala probably creates tight binding to the enzyme.
The structure of alanine racemase from Bacillus stearothermophilus (Geobacillus stearothermophilus) was determined by X-ray crystallography to a resolution of 1.9 A. The alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta barrel at the N terminus, and a C-terminal domain essentially composed of beta-strand. A model of the two domain structure is shown in Figure 2. The N-terminal domain is also found in the PROSC (proline synthetase co-transcribed bacterial homolog) family of proteins, which are not known to have alanine racemase activity. The pyridoxal 5'-phosphate (PLP) cofactor lies in and above the mouth of the alpha/beta barrel and is covalently linked via an aldimine linkage to a lysine residue, which is at the C terminus of the first beta-strand of the alpha/beta barrel.
Reaction mechanisms are difficult to fully prove by experiment. The traditional mechanism attributed to an alanine racemase reaction is that of a two-base mechanism with a PLP-stabilized carbanion intermediate. PLP is used as an electron sink stabilize the negative charge resulting from the deprotonation of the alpha carbon. The two based mechanism favors reaction specificity compared to a one base mechanism. The second catalytic residue is pre-positioned to donate a proton quickly after a carbanionic intermediate is formed and thus reduces the chance of alternative reactions occurring. There are two potential conflicts with this traditional mechanism, as identified by Watanabe et al. First, Arg219 forms a hydrogen bond with pyridine nitrogen of PLP. The arginine group has a pKa of about 12.6 and is therefore unlikely to protonate the pyridine. Normally in PLP reactions an acidic amino acid residue such as a carboxylic acid group, with a pKa of about 5, protonates the pyridine ring. The protonation of the pyridine nitrogen allows the nitrogen to accept additional negative charge. Therefore, due to the Arg219, the PLP-stabilized carbanion intermediate is less likely to form. Another problem identified was the need for another basic residue to return Lys39 and Tyr265 back to their protonated and unprotonated forms for L-alanine and vice versa for D-alanine. Watanabe et al. found no amino acid residues or water molecules, other than the carboxylate group of PLP-Ala, to be close enough (within 4.5A) to protonate or deprotonate Lys or Tyr. This is shown in Figure 3.
Based on the crystal structures of N-(5’-phosphopyridoxyl) L- alanine (PKP-L-Ala ( and N-(5’-phosphopyridoxyl) D-alanine (PLP-D-Ala)
Watanabe et al. proposed an alternative mechanism in 2002, as seen in the figure 4. In this mechanism the carboxylate oxygen atoms of PLP-Ala directly participates in catalysis by mediating proton transfer between Lys39 and Tyr265. The crystallization structure identified that the carboxylate oxygen of PLP-L-Ala to the OH of Tyr265 was only 3.6A and the carboxylate oxygen of PLP-L-Ala to the nitrogen of Lys39 was only 3.5A. Therefore, both were close enough to cause a reaction.
This mechanism is supported by mutations of Arg219. Mutations changing Arg219 to a carboxylate result in a quinonoid intermediate being detected whereas none was detected with arginine. The arginine intermediate has much more free energy, is more unstable, than the acidic residue mutants. The destabilization of the intermediate promotes specificity of the reaction,.
- Milligan Daniel L.; et al. (2007). "The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of D-Alanine". Journal of Bacteriology. 189 (22): 8381–8386. doi:10.1128/jb.01201-07. PMC . PMID 17827284.
- Abe, H; Yoshikawa, N; Sarower, M. G.; Okada, S (2005). "Physiological function and metabolism of free D-alanine in aquatic animals". Biological & Pharmaceutical Bulletin. 28 (9): 1571–7. doi:10.1248/bpb.28.1571. PMID 16141518.
- Watanabe, A., Yoshimura, T., Mikami, B., Hayashi, H., Kagamiyama, H., Esaki, N. (2002) Reaction Mechanism of Alanine Racemase from Bacillus stearothermophilus: X-ray crystallographic studies of the enzyme bound within -(5’-phosphopyridoxyl)alanine Journal of Biological Chemistry 277, 19166-19172.
- Stamper, G. F., Morollo, A. A., and Ringe, D. (1998) Biochemistry 37, 10438 –10445
- Morollo, A. A., Petsko, G. A., and Ringe, D. (1999) Biochemistry 38, 3293–3301
- Shaw, J. P., Petsko, G. A., and Ringe, D. (1997) Determination of the Structure of Alanine racemase from Bacillus stearothermophilus at 1.9-A Resolution Biochemistry 36, 1329–1342
- Toney, Michael D. (2004) Reaction specificity in pyridoxal phosphate enzymes, Archives of Biochemistry and Biophysics 433, 279-287.
- Sun S., Toney, M.D. (1998) Evidence for a Two-Base Mechanism Involving Tyrosine-265 from Arginine-219 Mutants of Alanine Racemase Biochemistry 38, 4058-4065
- Rubinstein, A., Major, D. T. (2010) Understanding Catalytic Specificity in Alanine Racemase from Quantum Mechanical Molecular Mechanical Simulations of the Arginine 210 Mutant Biochemistry 49, 3957-3963.
- MARR AG, WILSON PW (1954). "The alanine racemase of Brucella abortus". Arch. Biochem. Biophys. 49 (2): 424–33. doi:10.1016/0003-9861(54)90211-8. PMID 13159289.
- Wood WA (1955). "Amino acid racemases". Methods Enzymol. Methods in Enzymology. 2: 212–217. doi:10.1016/S0076-6879(55)02189-7. ISBN 9780121818029.
- WOOD WA, GUNSALUS IC (1951). "D-Alanine formation; a racemase in Streptococcus faecalis". J. Biol. Chem. 190 (1): 403–16. PMID 14841188.
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Alanine racemase, N-terminal domain Provide feedback
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Internal database links
|SCOOP:||ETF Orn_Arg_deC_N Orn_DAP_Arg_deC|
|Similarity to PfamA using HHSearch:||Orn_DAP_Arg_deC Orn_Arg_deC_N|
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR001608
Alanine racemase plays a role in providing the D-alanine required for cell wall biosynthesis by isomerising L-alanine to D-alanine.
The molecular structure of alanine racemase from Bacillus stearothermophilus was determined by X-ray crystallography to a resolution of 1.9 A [PUBMED:9063881]. The alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta barrel at the N terminus, and a C-terminal domain essentially composed of beta-strands. The pyridoxal 5'-phosphate (PLP) cofactor lies in and above the mouth of the alpha/beta barrel and is covalently linked via an aldimine linkage to a lysine residue, which is at the C terminus of the first beta-strand of the alpha/beta barrel.
This N-terminal domain is also found in the PROSC (proline synthetase co-transcribed bacterial homologue) family of proteins, which are not known to have alanine racemase activity.
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This large superfamily of TIM barrel enzymes all contain a common phosphate binding site. The phosphate is found in a variety of cofactors and ligands such as FMN [1,2].
The clan contains the following 59 members:4HFCP_synth Ala_racemase_N ALAD Aldolase AP_endonuc_2 BtpA CdhD ComA CutC DAHP_synth_1 DAHP_synth_2 DeoC DHDPS DHO_dh DHquinase_I DUF2090 DUF561 DUF692 DUF993 Dus F_bP_aldolase FMN_dh G3P_antiterm Glu_syn_central Glu_synthase His_biosynth HMGL-like IGPS IMPDH KDGP_aldolase Lys-AminoMut_A MtrH NanE NAPRTase NeuB NMO OAM_alpha OMPdecase Orn_Arg_deC_N Oxidored_FMN PcrB PdxJ PRAI PRMT5_TIM Pterin_bind QRPTase_C Radical_SAM RhaA Ribul_P_3_epim SOR_SNZ Tagatose_6_P_K TAL_FSA ThiC_Rad_SAM ThiG TIM TMP-TENI Trp_syntA UvdE UxuA
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|Number in seed:||317|
|Number in full:||12043|
|Average length of the domain:||219.60 aa|
|Average identity of full alignment:||20 %|
|Average coverage of the sequence by the domain:||65.04 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 26740544 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||19|
|Download:||download the raw HMM for this family|
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There are 5 interactions for this family. More...
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For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Ala_racemase_N domain has been found. There are 159 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.
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