328-50-7Relevant articles and documents
Coupling between D-3-phosphoglycerate dehydrogenase and D-2-hydroxyglutarate dehydrogenase drives bacterial L-serine synthesis
Zhang, Wen,Zhang, Manman,Gao, Chao,Zhang, Yipeng,Ge, Yongsheng,Guo, Shiting,Guo, Xiaoting,Zhou, Zikang,Liu, Qiuyuan,Zhang, Yingxin,Ma, Cuiqing,Tao, Fei,Xu, Ping
, p. E7574 - E7582 (2017)
L-Serine biosynthesis, a crucial metabolic process in most domains of life, is initiated by D-3-phosphoglycerate (D-3-PG) dehydrogenation, a thermodynamically unfavorable reaction catalyzed by D-3-PG dehydrogenase (SerA). D-2-Hydroxyglutarate (D-2-HG) is traditionally viewed as an abnormal metabolite associated with cancer and neurometabolic disorders. Here, we reveal that bacterial anabolism and catabolism of D-2-HG are involved in L-serine biosynthesis in Pseudomonas stutzeri A1501 and Pseudomonas aeruginosa PAO1. SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to D-2-HG, responsible for the major production of D-2-HG in vivo. SerA combines the energetically favorable reaction of D-2-HG production to overcome the thermodynamic barrier of D-3-PG dehydrogenation. We identified a bacterial D-2-HG dehydrogenase (D2HGDH), a flavin adenine dinucleotide (FAD)-dependent enzyme, that converts D-2-HG back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) are also essential in D-2-HG metabolism through their capacity to transfer electrons from D2HGDH. Furthermore, while the mutant with D2HGDH deletion displayed decreased growth, the defect was rescued by adding L-serine, suggesting that the D2HGDH is functionally tied to L-serine synthesis. Substantial flux flows through D-2-HG, being produced by SerA and removed by D2HGDH, ETF, and ETFQO, maintaining D-2-HG homeostasis. Overall, our results uncover that D-2-HG–mediated coupling between SerA and D2HGDH drives bacterial L-serine synthesis.
Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by l-amino acid deaminase
Hossain, Gazi Sakir,Shin, Hyun-Dong,Li, Jianghua,Wang, Miao,Du, Guocheng,Liu, Long,Chen, Jian
, p. 46149 - 46158 (2016)
l-Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from l-amino acids. In a previous study, a whole cell biocatalyst with the l-amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from l-glutamic acid, and the α-KG titer reached 12.79 g L-1 in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L-1 when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L-1 when monosodium glutamate was continuously fed at a constant rate of 6 g L-1 h-1 (from 4 to 23 h) with an initial concentration of 50 g L-1. Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min-1 to 241.8 μM min-1) and substrate affinity (the Km for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.
A new l-arginine oxidase engineered from l-glutamate oxidase
Yano, Yoshika,Matsuo, Shinsaku,Ito, Nanako,Tamura, Takashi,Kusakabe, Hitoshi,Inagaki, Kenji,Imada, Katsumi
, p. 1044 - 1055 (2021/04/14)
The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.
Enhanced nonradical catalytic oxidation by encapsulating cobalt into nitrogen doped graphene: highlight on interfacial interactions
Yu, Xiaoyong,Wang, Lijing,Wang, Xin,Liu, Hongzhi,Wang, Ziyuan,Huang, Yixuan,Shan, Guoqiang,Wang, Weichao,Zhu, Lingyan
supporting information, p. 7198 - 7207 (2021/03/29)
Supported metal catalysts are widely used for heterogeneous catalytic processes (e.g., Fenton-like reaction), but the mechanisms of interfacial processes are still ambiguous. Herein, unique nanocarbon based catalysts with Co nanoparticles encapsulated in
From a dimer to a monomer: Construction of a chimeric monomeric isocitrate dehydrogenase
Tian, Changqing,Wen, Bin,Bian, Mingjie,Jin, Mingming,Wang, Peng,Xu, Lei,Zhu, Guoping
, p. 2396 - 2407 (2021/10/29)
Many isocitrate dehydrogenases (IDHs) are dimeric enzymes whose catalytic sites are located at the intersubunit interface, whereas monomeric IDHs form catalytic sites with single polypeptide chains. It was proposed that monomeric IDHs were evolved from dimeric ones by partial gene duplication and fusion, but the evolutionary process had not been reproduced in laboratory. To construct a chimeric monomeric IDH from homo-dimeric one, it is necessary to reconstitute an active center by a duplicated region; to properly link the duplicated region to the rest part; and to optimize the newly formed protein surface. In this study, a chimeric monomeric IDH was successfully constructed by using homo-dimeric Escherichia coli IDH as a start point by rational design and site-saturation mutagenesis. The ~67 kDa chimeric enzyme behaved as a monomer in solution, with a Km of 61 μM and a kcat of 15 s?1 for isocitrate in the presence of NADP+ and Mn2+. Our result demonstrated that dimeric IDHs have a potential to evolve monomeric ones. The evolution of the IDH family was also discussed.
METHODS FOR IMPROVING YIELDS OF L-GLUFOSINATE
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Page/Page column 32, (2020/03/29)
Compositions and methods for the production of L-glufosinate are provided. The method involves converting racemic glufosinate to the L-glufosinate enantiomer or converting PRO to L-glufosinate in an efficient manner. In particular, the method involves the specific amination of PRO to L-glufosinate, using L-glutamate, racemic glutamate, or another amine source as an amine donor. PRO can be obtained by the oxidative deamination of D-glufosinate to PRO (2- oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid) or generated via chemical synthesis. PRO is then converted to L-glufosinate using a transaminase in the presence of an amine donor. When the amine donor donates an amine to PRO, L-glufosinate and a reaction by product are formed. Because the PRO remaining represents a yield loss of L-glufosinate, it is desirable to minimize the amount of PRO remaining in the reaction mixture. Degradation, other chemical modification, extraction, sequestration, binding, or other methods to reduce the effective concentration of the by-product, i.e., the corresponding alpha ketoacid or ketone to the chosen amine donor will shift the reaction equilibrium toward L-glufosinate, thereby reducing the amount of PRO and increasing the yield of L-glufosinate. Therefore, the methods described herein involve the conversion or elimination of the alpha ketoacid or ketone by-product to another product to shift the equilibrium towards L-glufosinate.
The pseudoalteromonas luteoviolacea L-amino acid oxidase with antimicrobial activity is a flavoenzyme
Andreo-Vidal, Andrés,Sanchez-Amat, Antonio,Campillo-Brocal, Jonatan C.
, (2019/01/03)
The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.
Characterization of aromatic aminotransferases from Ephedra sinica Stapf
Kilpatrick, Korey,Pajak, Agnieszka,Hagel, Jillian M.,Sumarah, Mark W.,Lewinsohn, Efraim,Facchini, Peter J.,Marsolais, Frédéric
, p. 1209 - 1220 (2016/04/26)
Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
Determinants of dual substrate specificity revealed by the crystal structure of homoisocitrate dehydrogenase from Thermus thermophilus in complex with homoisocitrate·Mg2+·NADH
Takahashi, Kento,Tomita, Takeo,Kuzuyama, Tomohisa,Nishiyama, Makoto
, p. 1688 - 1693 (2016/10/26)
HICDH (Homoisocitrate dehydrogenase) is a member of the β-decarboxylating dehydrogenase family that catalyzes the conversion of homoisocitrate to α-ketoadipate using NAD+ as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the α-aminoadipate pathway. Although typical HICDHs from fungi and yeast exhibit strict substrate specificities toward homoisocitrate (HIC), HICDH from a thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both HIC and isocitrate (IC) as substrates at similar efficiencies. We herein determined the crystal structure of the quaternary complex of TtHICDH with HIC, NADH, and Mg2+ ion at a resolution of 2.5??. The structure revealed that the distal carboxyl group of HIC was recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. Model structures were constructed for TtHICDH in complex with IC and also for HICDH from Saccharomyces cerevisiae (ScHICDH) in complex with HIC. TtHICDH recognized the distal carboxyl group of IC by Arg85 in the model. In ScHICDH, the distal carboxyl group of HIC was recognized by the side chains of Ser98 and Ser108 from one subunit and Asn208 from another subunit of a dimer unit. By contrast, in ScHICDH, which lacks an Arg residue at the position corresponding to Arg85 in TtHICDH, these residues may not interact with the distal carboxyl group of shorter IC. These results provide a molecular basis for the differences in substrate specificities between TtHICDH and ScHICDH.
Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases by 2-Thiohydantoin Compounds
Wu, Fangrui,Jiang, Hong,Zheng, Baisong,Kogiso, Mari,Yao, Yuan,Zhou, Chao,Li, Xiao-Nan,Song, Yongcheng
, p. 6899 - 6908 (2015/09/22)
Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can reduce α-ketoglutaric acid (α-KG) to d-2-hydroxyglutaric acid (D2HG). The resulting high concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases, to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for the initiation of these cancers. We report the synthesis, structure-activity relationships, enzyme kinetics, and binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which several compounds are potent inhibitors of mutant IDH1 with Ki as low as 420 nM. X-ray crystal structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of histone methylation, and suppress the proliferation of stem-like cancer cells in BT142 glioma with IDH1 R132H mutation.
