Preparation and characterisation of a Ni2+/Co2+-cyclam modified mesoporous cellular foam for the specific immobilisation of His6-alanine racemase
Darragh GaffneyNoreldeen H. AbdallahJakki C. CooneyFathima LaffirKarim Engelmark CassimjeePer BerglundUlf HanefeldEdmond Magner
12
Citation
36
Reference
10
Related Paper
Citation Trend
Keywords:
Cyclam
Alanine
During 2 min incubations of Trypanosoma gambiense (bloodstream form) with [U- 14 C]glucose (1 mM) over 60% of absorbed label was detected in free alanine. In the presence of 12·5 mM unlabelled alanine, the amount of alanine synthesized from glucose was reduced by less than 10%. These data support previous observations on the high level of transaminase activity in African human trypanosomes. Alanine, aspartate and glutamate were metabolized to various other free amino acids whereas a significant amount of label derived from [ 14 C]arginine could not be accounted for by amino acid assay. The sulphur containing amino acids, cysteic acid and taurine, were apparently synthesized from alanine, glutamate and arginine. The significance of these syntheses is poorly understood. Following incubations of trypanosomes for 2 min in exogenous amino acids the internal free pool became imbalanced due to accumulation and metabolism of the substrate amino acid. Evidence obtained indicated that the level of free endogenous glutamate may be rate limiting for the glutamate-pyruvate transaminase system.
Cysteic acid
Alanine
Transaminase
Cite
Citations (15)
Abstract Both isomeric forms of alanine play a crucial role in bacterial growth and viability; the L‐isomer of this amino acid is one of the building blocks for protein synthesis, and the D‐isomer is incorporated into the bacterial cell wall. Despite a long history of genetic manipulation of Bacillus subtilis using auxotrophic markers, the genes involved in alanine metabolism have not been characterized fully. In this work, we genetically characterized the major enzymes involved in B . subtilis alanine biosynthesis and identified an alanine permease, AlaP (YtnA), which we show has a major role in the assimilation of D‐alanine from the environment. Our results provide explanations for the puzzling fact that growth of B . subtilis does not result in the significant accumulation of extracellular D‐alanine. Interestingly, we find that in B . subtilis , unlike E . coli where multiple enzymes have a biochemical activity that can generate alanine, the primary synthetic enzyme for alanine is encoded by alaT , although a second gene, dat , can support slow growth of an L‐alanine auxotroph. However, our results also show that Dat mediates the synthesis of D‐alanine and its activity is influenced by the abundance of L‐alanine. This work provides valuable insights into alanine metabolism that suggests that the relative abundance of D‐ and L‐alanine might be linked with cytosolic pool of D and L‐glutamate, thereby coupling protein and cell envelope synthesis with the metabolic status of the cell. The results also suggest that, although some of the purified enzymes involved in alanine biosynthesis have been shown to catalyze reversible reactions in vitro, most of them function unidirectionally in vivo.
Alanine
Cite
Citations (20)
UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli was overexpressed more than 600-fold and purified to near homogeneity. The purified enzyme was found to ligate L-alanine, L-serine, and glycine, as well as the nonnatural amino acid beta-chloro-L-alanine, to UDP-N-acetylmuramic acid. On the basis of (i) the specificity constants of the enzyme determined for L-alanine, L-serine, and glycine and (ii) the levels of these amino acids in the intracellular pool, it was calculated that the rates of incorporation of L-serine and glycine into peptidoglycan precursor metabolites could maximally amount to 0.1 and 0.5%, respectively, of the rate of L-alanine incorporation.
Alanine
Cite
Citations (24)
1. Factors regulating the release of alanine and glutamine in vivo were investigated in starved rats by removing the liver from the circulation and monitoring blood metabolite changes for 30 min. 2. Alanine and glutamine were the predominant amino acids released into the circulation in this preparation. 3. Dichloroacetate, an activator of pyruvate dehydrogenase, inhibited net alanine release: it also interfered with the metabolism of the branched-chain amino acids valine, leucine and isoleucine. 4. L-Cycloserine, an inhibitor of alanine aminotransferase, decreased alanine accumulation by 80% after functional hepatectomy, whereas methionine sulphoximine, an inhibitor of glutamine synthetase, decreased glutamine accumulation by the same amount. 5. It was concluded that: (a) the alanine aminotransferase and the glutamine synthetase pathways respectively were responsible for 80% of the alanine and glutamine released into the circulation by the extrasplanchnic tissues, and extrahepatic proteolysis could account for a maximum of 20%; (b) alanine formation by the peripheral tissues was dependent on availability of pyruvate and not of glutamate; (c) glutamate availability could influence glutamine formation subject, possibly, to renal control.
Alanine
Isoleucine
Cite
Citations (38)
Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, gamma-aminobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by l-methionine-d,l-sulfoximine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism.
Alanine
Amino acid synthesis
Azaserine
Aspartic acid
Chara
Glutamic acid
Isoleucine
Cite
Citations (19)
Abstract The macrocycles (Ib), (Va) and (Vb), in which a methyl group is introduced on an N atom of the parent cyclam (Ic) or the isocyclam (Vc), are prepared as shown in the scheme.
Cyclam
Cite
Citations (0)
Four alanine aminotransferases (AlaATs) are expressed in Medicago truncatula. In adult plants, two genes encoding mitochondrial isoforms m-AlaAT and alanine–glyoxylate aminotransferase (AGT), catalysing, respectively, reversible reactions of alanine/oxoglutarate↔glutamate/pyruvate and alanine/glyoxylate↔glycine/pyruvate, were expressed in roots, stems, and leaves. A gene encoding a cytosolic (c-AlaAT) isoform, catalysing the same reaction as m-AlaAT, was expressed specifically in leaves, while a gene encoding an isoform involved in branched chain amino acid metabolism was expressed in stems and roots. In young seedlings, only m-AlaAT and AGT were expressed in embryo axes. In hypoxic embryo axes, the amounts of transcript and putative protein of m-AlaAT (EC 2.6.1.2) increased while those of AGT (EC 2.6.1.44) decreased and in vivo enzyme activities changed as revealed by [15N]alanine and [15N]glutamate labelling. Under hypoxia, m-AlaAT catalysed only alanine synthesis while glutamate synthesis using alanine as amino donor was inhibited. As a result, alanine accumulated as the major amino acid in hypoxic seedlings instead of asparagine, in agreement with the involvement of the fermentative AlaAT pathway in hypoxia tolerance. Regulation of m-AlaAT at both the transcriptional and post-translational levels allowed for an increase in gene expression and orientation of the activity of the product of its transcription towards alanine synthesis under hypoxia. Labelling experiments showed that glycine synthesis occurred at the expense of either alanine or glutamate as amino donor, indicating that a glutamate–glyoxylate aminotransferase was operating together with AGT in Medicago truncatula seedlings. Both enzymes seemed to be inhibited by hypoxia, resulting in a very low amount of glycine in hypoxic seedlings.
Alanine
Medicago truncatula
Cite
Citations (110)
Mesoporous materials containing bridged cyclam moieties inside the framework were prepared by using a neutral templating route. Quantitative formation of bridged CuII-cyclam complexes was obtained by the direct incorporation of CuCl2 inside the hybrid material, showing thus the complete accessibility of cyclam moieties located inside the framework. Grafting of a metal-N-triethoxysilylpropylcyclam complex inside the channel pores followed by the incorporation of another metal salt into the framework gave rise to an hybrid material containing two strongly chelated metal salts, one located inside the framework, the other in the channel pores.
Cyclam
Hybrid material
Cite
Citations (62)
Abstract The complexing of Cu(II) by the title ligand L is investigated by potentiometric titration and by UV/VIS spectroscopy.
Cyclam
Cite
Citations (0)