Aspartate racemase

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aspartate racemase
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EC no. 5.1.1.13
CAS no. 37237-56-2
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In enzymology, an aspartate racemase (EC 5.1.1.13) is an enzyme that catalyzes the following chemical reaction:

Contents

L-aspartate D-aspartate

This enzyme belongs to the family of isomerases, specifically those racemases and epimerases acting on amino acids and amino acid derivatives, including glutamate racemase, proline racemase, and diaminopimelate epimerase.

The systematic name of this enzyme class is aspartate racemase. Other names in common use include D-aspartate racemase, and McyF. [1]

Discovery

Aspartate racemase was first discovered in the gram-positive bacteria Streptococcus faecalis by Lamont et al. in 1972. [2] It was then determined that aspartate racemase also racemizes L-alanine around half as quickly as it does L-aspartate, but does not show racemase activity in the presence of L-glutamate.

Structure

The crystallographic structure of bacterial aspartate racemase has been solved in Pyrococcus horikoshii OT3, [3] Escherichia coli , Microcystis aeruginosa , and Picrophilus torridus DSM 9790 .

Homodimer

Homodimeric aspartate racemase isolated from P. horikoshii, viewed at an angle showing the binding pocket. The different colors refer to the different monomers of the complex. Aspartate racemase cleft view.png
Homodimeric aspartate racemase isolated from P. horikoshii, viewed at an angle showing the binding pocket. The different colors refer to the different monomers of the complex.

In most bacteria for which the structure is known, aspartate racemase exists as a homodimer, [3] where each subunit has a molecular weight of approximately 25 kDa. The complex consists primarily of alpha helices, and additionally features a Rossmann fold in the center of the dimer. The catalytic pocket lies at the cleft formed by the intersection of the two domains. A citrate molecule can fit inside the binding pocket, leading to a contraction of the cleft to make the "closed form" of aspartate racemase. [4]

Two highly conserved cysteine residues are suggested to be responsible for the interconversion of L-aspartate and D-aspartate. [3] These cysteine residues lie 3–4 angstroms away from the α-carbon of aspartate. Site-directed mutagenesis studies showed that the mutation of the upstream cysteine residue to serine resulted in complete loss of racemization activity, while the same mutation in the downstream cysteine residue resulted in retention of 10–20% racemization activity. [4] However, mutation of the acid residue glutamate, which stabilizes the downstream cysteine residue, resulted in complete loss of racemization activity. Up to 9 other residues are known to interact with and stabilize the isomers of aspartate through hydrogen bonding or hydrophobic interactions. [5] [4]

In E. coli, one of the active cysteine residues is substituted for a threonine residue, allowing for much greater substrate promiscuity. [5] Notably, aspartate racemase in E. coli is also able to catalyze the racemization of glutamate.

Monomer

In 2004, an aspartate racemase was discovered in Bifidobacterium bifidum as a 27 kDa monomer. [6] This protein shares nearly identical enzymological properties with homodimeric aspartate racemase isolated from Streptococcus thermophilus , but has the added characteristic that its thermal stability increases significantly in the presence of aspartate.

Reaction mechanism

Aspartate racemase catalyzes the following reaction:

Aspartate racemase general reaction.png

Aspartate racemase can accept either L-aspartate or D-aspartate as substrates.

Amino acid racemization is carried out by two dominant mechanisms: one-base mechanisms and two-base mechanisms. [7] In one-base mechanisms, a proton acceptor abstracts the α-hydrogen from the substrate amino acid to form a carbanion intermediate until reprotonated at the other face of the α-carbon. Racemases dependent on pyridoxal-5-phosphate (PLP) typically leverage one-base mechanisms. In the two-base mechanism, an alpha hydrogen is abstracted by a base on one face of the amino acid while another protonated base concertedly donates its hydrogen onto the other face of the amino acid.

PLP-independent mechanism

Aspartate racemases in bacteria function in the absence of PLP, suggesting a PLP-independent mechanism. [5] A two-base mechanism is supported in the literature, carried out by two thiol groups:

Aspartate racemization mechanism two base.png

Other PLP-independent isomerases in bacteria include glutamate racemase, proline racemase, and hydroxyproline-2-epimerase.

PLP-dependent mechanism

Mammalian aspartate racemase, in contrast with bacterial aspartate racemase, is a PLP-dependent enzyme. The exact mechanism is unknown, but it is hypothesized to proceed similarly to mammalian serine racemase as below:

Aspartate racemase PLP-dependent mechanism.png

Inhibition

General inhibitors for cysteine residues have shown to be effective agents against monomeric aspartate racemase. [2] [5] N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) both inhibit monomeric aspartate racemase at 1mM.

Function

Metabolism of D-aspartate

One of the primary functions of aspartate racemase in bacteria is the metabolism of D-aspartate. [8] [9] The beginning of D-aspartate metabolism is its conversion to L-alanine. First, D-aspartate is isomerized to L-aspartate by aspartate racemase, followed by decarboxylation to form L-alanine. [9]

Metabolism of D-aspartate to L-alanine.png

Peptidoglycan synthesis

D-amino acids are common within the peptidoglycan of bacteria. [10] In Bifidobacterium bifidum , D-aspartate is formed from L-aspartate via aspartate racemase and used as a cross-linker moiety in the peptidoglycan. [6]

Mammalian neurogenesis

Aspartate racemase is highly expressed in the brain, the heart, and the testes of mammals, all tissues in which D-aspartate is present. [11] D-aspartate is abundant in the embryonic brain, but falls during postnatal development. Retrovirus-mediated expression of short hairpin RNA complementary to aspartate racemase in newborn neurons of the adult hippocampus led to defects in dendritic development and empaired survival of the newborn neurons, suggesting that aspartate racemase may modulate adult neurogenesis in mammals. [12]

Evolution

Phylogenetic analysis shows that PLP-dependent animal aspartate racemases are in the same family as PLP-dependent animal serine racemases, and the genes encoding them share a common ancestor. [13] Aspartate racemases in animals have independently evolved from serine racemases through amino acid substitutions, namely, the introduction of three consecutive serine residues. [14] Serine racemases isolated from Saccoglossus kowalevskii also show both high aspartate and glutamate racemization activity. [15]

Related Research Articles

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Methionine is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical role in the metabolism and health of many species, including humans. It is encoded by the codon AUG.

Serine is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group, a carboxyl group, and a side chain consisting of a hydroxymethyl group, classifying it as a polar amino acid. It can be synthesized in the human body under normal physiological circumstances, making it a nonessential amino acid. It is encoded by the codons UCU, UCC, UCA, UCG, AGU and AGC.

<span class="mw-page-title-main">Pyridoxal phosphate</span> Active form of vitamin B6

Pyridoxal phosphate (PLP, pyridoxal 5'-phosphate, P5P), the active form of vitamin B6, is a coenzyme in a variety of enzymatic reactions. The International Union of Biochemistry and Molecular Biology has catalogued more than 140 PLP-dependent activities, corresponding to ~4% of all classified activities. The versatility of PLP arises from its ability to covalently bind the substrate, and then to act as an electrophilic catalyst, thereby stabilizing different types of carbanionic reaction intermediates.

<span class="mw-page-title-main">Aspartate transaminase</span> Enzyme involved in amino acid metabolism

Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or (serum) glutamic oxaloacetic transaminase, is a pyridoxal phosphate (PLP)-dependent transaminase enzyme that was first described by Arthur Karmen and colleagues in 1954. AST catalyzes the reversible transfer of an α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, red blood cells and gall bladder. Serum AST level, serum ALT level, and their ratio are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels.

In molecular biology, biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. This process often consists of metabolic pathways. Some of these biosynthetic pathways are located within a single cellular organelle, while others involve enzymes that are located within multiple cellular organelles. Examples of these biosynthetic pathways include the production of lipid membrane components and nucleotides. Biosynthesis is usually synonymous with anabolism.

<span class="mw-page-title-main">Amino acid synthesis</span> The set of biochemical processes by which amino acids are produced

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<span class="mw-page-title-main">Serine hydroxymethyltransferase</span>

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP) (Vitamin B6) dependent enzyme (EC 2.1.2.1) which plays an important role in cellular one-carbon pathways by catalyzing the reversible, simultaneous conversions of L-serine to glycine and tetrahydrofolate (THF) to 5,10-Methylenetetrahydrofolate (5,10-CH2-THF). This reaction provides the largest part of the one-carbon units available to the cell.

<span class="mw-page-title-main">Serine dehydratase</span>

Serine dehydratase or L-serine ammonia lyase (SDH) is in the β-family of pyridoxal phosphate-dependent (PLP) enzymes. SDH is found widely in nature, but its structural and properties vary among species. SDH is found in yeast, bacteria, and the cytoplasm of mammalian hepatocytes. SDH catalyzes is the deamination of L-serine to yield pyruvate, with the release of ammonia.

<span class="mw-page-title-main">Cystathionine gamma-lyase</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">Transsulfuration pathway</span>

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<span class="mw-page-title-main">Alanine racemase</span>

In enzymology, an alanine racemase is an enzyme that catalyzes the chemical reaction

In enzymology, an amino-acid racemase is an enzyme that catalyzes the chemical reaction

In enzymology, an arginine racemase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Diaminopimelate epimerase</span>

In enzymology, a diaminopimelate epimerase is an enzyme that catalyzes the chemical reaction

In enzymology, glutamate racemase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Cystathionine beta-lyase</span> Enzyme

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<span class="mw-page-title-main">Methionine gamma-lyase</span>

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<span class="mw-page-title-main">Arginine decarboxylase</span>

The enzyme Acid-Induced Arginine Decarboxylase (AdiA), also commonly referred to as arginine decarboxylase, catalyzes the conversion of L-arginine into agmatine and carbon dioxide. The process consumes a proton in the decarboxylation and employs a pyridoxal-5'-phosphate (PLP) cofactor, similar to other enzymes involved in amino acid metabolism, such as ornithine decarboxylase and glutamine decarboxylase. It is found in bacteria and virus, though most research has so far focused on forms of the enzyme in bacteria. During the AdiA catalyzed decarboxylation of arginine, the necessary proton is consumed from the cell cytoplasm which helps to prevent the over-accumulation of protons inside the cell and serves to increase the intracellular pH. Arginine decarboxylase is part of an enzymatic system in Escherichia coli, Salmonella Typhimurium, and methane-producing bacteria Methanococcus jannaschii that makes these organisms acid resistant and allows them to survive under highly acidic medium.

<span class="mw-page-title-main">Diaminopimelate decarboxylase</span>

The enzyme diaminopimelate decarboxylase (EC 4.1.1.20) catalyzes the cleavage of carbon-carbon bonds in meso 2,6 diaminoheptanedioate to produce CO2 and L-lysine, the essential amino acid. It employs the cofactor pyridoxal phosphate, also known as PLP, which participates in numerous enzymatic transamination, decarboxylation and deamination reactions.

D-Amino acids are amino acids where the stereogenic carbon alpha to the amino group has the D-configuration. For most naturally-occurring amino acids, this carbon has the L-configuration. D-Amino acids are occasionally found in nature as residues in proteins. They are formed from ribosomally-derived D-amino acid residues.

References

  1. ↑ Sielaff H, Dittmann E, Tandeau De Marsac N, Bouchier C, Von Döhren H, Börner T, Schwecke T (August 2003). "The mcyF gene of the microcystin biosynthetic gene cluster from Microcystis aeruginosa encodes an aspartate racemase". The Biochemical Journal. 373 (Pt 3): 909–16. doi:10.1042/BJ20030396. PMC   1223527 . PMID   12713441.
  2. 1 2 Lamont HC, Staudenbauer WL, Strominger JL (August 1972). "Partial purification and characterization of an aspartate racemase from Streptococcus faecalis". The Journal of Biological Chemistry. 247 (16): 5103–6. doi: 10.1016/S0021-9258(19)44944-2 . PMID   4626916.
  3. 1 2 3 Liu L, Iwata K, Kita A, Kawarabayasi Y, Yohda M, Miki K (May 2002). "Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization". Journal of Molecular Biology. 319 (2): 479–89. doi:10.1016/S0022-2836(02)00296-6. PMID   12051922.
  4. 1 2 3 Cao DD, Zhang CP, Zhou K, Jiang YL, Tan XF, Xie J, et al. (July 2019). "Structural insights into the catalysis and substrate specificity of cyanobacterial aspartate racemase McyF". Biochemical and Biophysical Research Communications. 514 (4): 1108–1114. doi:10.1016/j.bbrc.2019.05.063. PMID   31101340.
  5. 1 2 3 4 Fischer C, Ahn YC, Vederas JC (December 2019). "Catalytic mechanism and properties of pyridoxal 5'-phosphate independent racemases: how enzymes alter mismatched acidity and basicity". Natural Product Reports. 36 (12): 1687–1705. doi:10.1039/c9np00017h. PMID   30994146.
  6. 1 2 Yamashita T, Ashiuchi M, Ohnishi K, Kato S, Nagata S, Misono H (December 2004). "Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum". European Journal of Biochemistry. 271 (23–24): 4798–803. doi: 10.1111/j.1432-1033.2004.04445.x . PMID   15606767.
  7. ↑ Yamauchi T, Choi SY, Okada H, Yohda M, Kumagai H, Esaki N, Soda K (September 1992). "Properties of aspartate racemase, a pyridoxal 5'-phosphate-independent amino acid racemase". The Journal of Biological Chemistry. 267 (26): 18361–4. doi: 10.1016/S0021-9258(19)36969-8 . PMID   1526977.
  8. ↑ Henríquez T, Salazar JC, Marvasi M, Shah A, Corsini G, Toro CS (2020). "SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000". PLOS ONE. 15 (1): e0228178. Bibcode:2020PLoSO..1528178H. doi: 10.1371/journal.pone.0228178 . PMC   6980539 . PMID   31978153.
  9. 1 2 Markovetz AJ, Cook WJ, Larson AD (August 1966). "Bacterial metabolism of d-aspartate involving racemization and decarboxylation". Canadian Journal of Microbiology. 12 (4): 745–51. doi:10.1139/m66-101. PMID   5969337.
  10. ↑ Lam H, Oh DC, Cava F, Takacs CN, Clardy J, de Pedro MA, Waldor MK (September 2009). "D-amino acids govern stationary phase cell wall remodeling in bacteria". Science. 325 (5947): 1552–5. Bibcode:2009Sci...325.1552L. doi:10.1126/science.1178123. PMC   2759711 . PMID   19762646.
  11. ↑ Ohide H, Miyoshi Y, Maruyama R, Hamase K, Konno R (November 2011). "D-Amino acid metabolism in mammals: biosynthesis, degradation and analytical aspects of the metabolic study". Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 879 (29): 3162–8. doi:10.1016/j.jchromb.2011.06.028. PMID   21757409.
  12. ↑ Kim PM, Duan X, Huang AS, Liu CY, Ming GL, Song H, Snyder SH (February 2010). "Aspartate racemase, generating neuronal D-aspartate, regulates adult neurogenesis". Proceedings of the National Academy of Sciences of the United States of America. 107 (7): 3175–9. Bibcode:2010PNAS..107.3175K. doi: 10.1073/pnas.0914706107 . PMC   2840285 . PMID   20133766.
  13. ↑ Uda K, Abe K, Dehara Y, Mizobata K, Sogawa N, Akagi Y, et al. (February 2016). "Distribution and evolution of the serine/aspartate racemase family in invertebrates". Amino Acids. 48 (2): 387–402. doi:10.1007/s00726-015-2092-0. PMID   26352274. S2CID   17216091.
  14. ↑ Uda K, Abe K, Dehara Y, Mizobata K, Edashige Y, Nishimura R, et al. (October 2017). "Triple serine loop region regulates the aspartate racemase activity of the serine/aspartate racemase family". Amino Acids. 49 (10): 1743–1754. doi:10.1007/s00726-017-2472-8. PMID   28744579. S2CID   3707632.
  15. ↑ Uda K, Ishizuka N, Edashige Y, Kikuchi A, Radkov AD, Moe LA (June 2019). "Cloning and characterization of a novel aspartate/glutamate racemase from the acorn worm Saccoglossus kowalevskii". Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 232: 87–92. doi:10.1016/j.cbpb.2019.03.006. PMID   30902582.
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