302-84-1Relevant articles and documents
Catalytic mechanism of serine racemase from Dictyostelium discoideum
Ito, Tomokazu,Maekawa, Motoki,Hayashi, Shuhei,Goto, Masaru,Hemmi, Hisashi,Yoshimura, Tohru
, p. 1073 - 1084 (2013)
The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.
Insights into the mechanism of pseudomonas dacunhae aspartate β-decarboxylase from rapid-scanning stopped-flow kinetics
Phillips, Robert S.,Lima, Santiago,Khristoforov, Roman,Sudararaju, Bakthavatsalam
, p. 5066 - 5073 (2010)
The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate β-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium l-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5′-phosphate and oxaloacetate or pyruvate. After consumption of the l-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate. In contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-β-hydroxy-dl-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of ～1 or 0.006 s-1, respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of l-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. It is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5′-phosphate Schiff base and increases the pKa, resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM l-Asp also results in the formation of the 325 nm complex, but with an apparent rate constant of 0.2 s-1, at least 5000-fold slower than the rate of wild-type ABDC. In contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role in modulating the pKa of the pyridoxal 5′-phosphate complexes during catalysis.
Metal ion dependency of serine racemase from Dictyostelium discoideum
Ito, Tomokazu,Murase, Hirotaka,Maekawa, Motoki,Goto, Masaru,Hayashi, Shuhei,Saito, Hajime,Maki, Masatoshi,Hemmi, Hisashi,Yoshimura, Tohru
, p. 1567 - 1576 (2012)
d-Serine is known to act as an endogenous co-Agonist of the N-methyl-d-Aspartate receptor in the mammalian brain and is endogenously synthesized from l-serine by Apyridoxal 5′-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the d-serine metabolism, such as serine racemase, d-Amino acid oxidase, and d-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of d-serine such as Arole in cell development. As part of the elucidation of the role of d-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na+ in addition to Mg2+ and Ca2+, which are well-known activators for the mammalian serine racemase. Mg2+ or Na+ binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg2+ and Na+ were determined to be 1.2 μM and 2.2 mM, respectively. In the l-serine dehydrase reaction, Mg2+ and Na + enhanced the k cat value without changing the K m value. Alanine mutation of the residues E207 and D213, which correspond to the Mg2+-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg2+- and Na+-dependent stimulation. These results suggest that Mg2+ and Na+ share the common metal ion-binding site.
Site-directed mutagenesis of rice serine racemase: Evidence that Glu219 and Asp225 mediate the effects of Mg2+ on the activity
Gogami, Yoshitaka,Kobayashi, Ai,Ikeuchi, Toshihiko,Oikawa, Tadao
, p. 1579 - 1590 (2010)
We succeeded in constructing the Glu219Ala/Asp225Ala (i.e., E219A/D225A) serine racemase (SerR) by site-directed mutagenesis, and the effects of Mg 2+ on the catalytic efficiency and the structure were compared between the E219A/D225A-SerR and the wild-type protein. This is the first example of a serine racemase whose amino acid residues in the Mg 2+-binding site were replaced with other amino acids by site-directed mutagenesis. Neither the serine racemase nor the dehydratase activities of the E219A/D225A-SerR were affected by the addition of Mg2+, and Glu219 and Asp225 of the SerR are the essential amino acid residues for Mg2+ to affect both kinds of enzyme activities. Therefore, Glu219 and Asp225 mediate the effects of Mg2+ on the activity and are important for the SerR to form the Mg2+-binding site. Judging from the difference of the Keq values between the E219A/D225A-SerR and the SerR, Mg2+ might affect the equilibrium states in the racemase reaction. The fluorescence quenching analysis of the E219A/D225A-SerR showed that Mg2+ bound to Glu219 and Asp225 of the SerR probably causes a conformational change in the ternary structure of the SerR.
Direct monitoring of biocatalytic deacetylation of amino acid substrates by1H NMR reveals fine details of substrate specificity
De Cesare, Silvia,McKenna, Catherine A.,Mulholland, Nicholas,Murray, Lorna,Bella, Juraj,Campopiano, Dominic J.
supporting information, p. 4904 - 4909 (2021/06/16)
Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that thel-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range ofN-acyl-amino acid substrates. This activity was revealed by1H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.
Dynamic Kinetic Resolution for Asymmetric Synthesis of L-Noncanonical Amino Acids from D-Ser Using Tryptophan Synthase and Alanine Racemase
Yu, Jinhai,Li, Jing,Gao, Xia,Zeng, Shuiyun,Zhang, Hongjuan,Liu, Junzhong,Jiao, Qingcai
, p. 6618 - 6625 (2019/11/03)
L-Ser is often used to synthesize some significant l-noncanonical α-amino acids(l-ncAAs), which are the prevalent intermediates and precursors for functional synthetic compounds. In this study, threonine aldolase from Escherichia coli k-12 MG1655 has been used to synthesize l-Ser. In contrast to the maximum catalytic capacity (20 g/L) for l-threonine aldolase(LTA), d-Ser was synthesized with high yield (240 g/L) from cheap Gly and paraformaldehyde using d-threonine aldolase (DTA) from Arthrobacter sp ATCC. In order to fully utilize d-Ser and expand the resource of l-Ser, a dynamic kinetic resolution system was constructed to convert d/dl-Ser to l-Ser through combining alanine racemase (Alr) from Bacillus subtilis with l-tryptophan synthase (TrpS) from Escherichia coli k-12 MG1655, and l-ncAAs including l-Trp and l-Cys derivatives were synthesized with excellent enantioselectivity and in high yields. The results indicated l-ncAAs could be efficiently synthesized from d-Ser using this original and green dynamic kinetic resolution system, and the reliable l-Ser resource has been established from simple and achiral substrates.
Preparation and purification method of amino acid compound
-
Paragraph 0058; 0059; 0072; 0073; 0074; 0075, (2018/06/21)
The invention relates to the field of industrial organic synthesis, in particular to a preparation and purification method of an amino acid compound. The method comprises the following steps that (1)alpha-amino nitrile compounds or hydantoin compounds or mixtures thereof are heated to react to obtain alpha-amino acid salt under the condition that volatile alkali and a suitable solvent exist; (2)after the alpha-amino acid salt obtained in step (1) is distilled, the alpha-amino acid salt is recrystallized in an organic solvent to obtain the alpha-amino acid compound. According to the method, reaction conditions are mild, materials can be recycled, and introduction of metal ions and use of ammonium carbonate salt are avoided, so that post-treatment is simple and no waste salt is generated.
A α - amino acid compound synthesis and purification method
-
Paragraph 0071; 0072; 0074, (2018/05/16)
The invention relates to a synthesis and purification method for an alpha-amino acid compound. The synthesis and purification method is characterized by comprising the following steps: (1) adding substituted alpha-amino nitrile or a substituted hydantoin-based compound into alkali M(OH)x or metal oxide MxO, adding water or an alcohol and water mixed solvent, and heating for reaction to obtain alpha-amino acid salt; (2) adding ammonium carbonate or ammonium bicarbonate or introducing carbon dioxide into the solution in the step (1), separating to obtain filter liquor and precipitates MxHyCO3, performing reduced pressure concentration on the filter liquor, and recrystallizing in an alcohol solvent to obtain the alpha-amino acid compound (I). The synthesis and purification method for the alpha-amino acid compound is simple, the yield and purity of the obtained alpha-amino acid compound are high; furthermore, recycling utilization and cleaning production of materials can be realized; the synthesis and purification method is especially suitable for synthesis of the alpha-amino acid compound with high water solubility.
Degradation of complexons derived from succinic acid under UV radiation
Smirnova,Khizhnyak,Nikol’skii,Khalyapina, Ya. M.,Pakhomov
, p. 507 - 511 (2017/08/02)
The destruction of complexons derived from succinic acid under the action of UV radiation was studied. IR spectroscopy, thin-layer paper chromatography, and complexometric titration were used to determine the destruction products of these complexons. It was found that the complexons decompose under UV irradiation substantially more easily than ethylenediaminetetraacetic acid does, and the products of their decomposition can undergo a biological utilization under natural conditions. The data obtained in the study make it possible to choose, instead of ethylenediaminetetraacetic acid, ligands that will be nearly fully destructible in the light without deteriorating the ecology.
Amide bond hydrolysis in peptides and cyclic peptides catalyzed by a dimeric Zr(iv)-substituted Keggin type polyoxometalate
Ly, Hong Giang T.,Absillis, Gregory,Parac-Vogt, Tatjana N.
, p. 10929 - 10938 (2013/09/12)
Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the presence of the dimeric zirconium(iv)-substituted Keggin type polyoxometalate (Et2NH2)8[{α -PW11O39Zr(μ-OH)(H2O)}2] ·7H2O (1) were performed by a combination of 1H, 13C and 31P NMR spectroscopy. The observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10-7 s-1 and 4.44 × 10-7 s -1 respectively, representing a significant acceleration as compared to the uncatalyzed reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile with the fastest hydrolysis observed in the pD range of 5.5-6.0. Interaction of 1 with Gly-Ser and Gly-Gly via their amine nitrogen and amide oxygen was proven by 13C NMR spectroscopy. The effective hydrolysis of Gly-Ser in the presence of 1 is most likely a combination of the polarization of the amide oxygen due to its binding to the Zr(iv) ion in 1 and the intramolecular attack of the Ser hydroxyl group on the amide carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant was also examined. The solution structure of 1 was investigated by means of 31P NMR spectroscopy, revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution of 1 was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence and in the absence of the dipeptides. The Royal Society of Chemistry 2013.
