The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 184.108.40.206) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.
"More specifically, they use the NAD+-dependent non-metalloenzyme ornithine cyclodeaminase (OCD) to directly convert the amino acid L-ornithine (L-Orn), itself also an intermediate along the above “Arg-pathway”, to proline . Thus, in contrast to the two general pathways discussed above in which the actual cyclization step is done without enzymatic participation, the mechanism of OCD includes the cyclization of a linear intermediate to a cyclic product (Scheme I) [9,29,30]. Furthermore, it not only produces L-proline stereospecifically and without additional enzymes being involved, but does so in an irreversible fashion [9,31]. "
[Show abstract][Hide abstract] ABSTRACT: Ornithine cyclodeaminase (OCD) is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD) and a hybrid quantum mechanics/molecular mechanics (QM/MM) method in the ONIOM formalism, the catalytic mechanism of OCD has been examined. The rate limiting step is calculated to be the initial step in the overall mechanism: hydride transfer from the L-ornithine’s Cα–H group to the NAD+ cofactor with concomitant formation of a Cα=NH2
+ Schiff base with a barrier of 90.6 kJ mol−1. Importantly, no water is observed within the active site during the MD simulations suitably positioned to hydrolyze the Cα=NH2
+ intermediate to form the corresponding carbonyl. Instead, the reaction proceeds via a non-hydrolytic mechanism involving direct nucleophilic attack of the δ-amine at the Cα-position. This is then followed by cleavage and loss of the α-NH2 group to give the Δ1-pyrroline-2-carboxylate that is subsequently reduced to L-proline.
International Journal of Molecular Sciences 12/2012; 13(10):12994-3011. DOI:10.3390/ijms131012994 · 2.86 Impact Factor
"Accumulation of L-alanine is also reported in ancestral bacterial anaerobes, as it is readily synthesized from glucose through reductive amination of pyruvate by alanine dehydrogenase (ALAD) [EC 220.127.116.11] as in the case with lactic acid bacteria (Kleerebezemab et al. 2000), E. coli (Lee et al. 2004) and sulfur-metabolizing strict anaerobes such as archaeon Archaeoglobus fulgidus (Gallagher et al. 2004). The known capability of anaerobes to produce L-alanine has broad application in pharmaceutical industry (Kleerebezemab et al. 2000; Wada et al. 2007) where it is manufactured from the carbon in glucose as a by-product of energy production in the absence of O 2 (Hashimoto and Katsumata 1999). "
[Show abstract][Hide abstract] ABSTRACT: Tumor cells have a high tolerance for acidic and hypoxic microenvironments, also producing abundant lactic acid through accelerated glycolysis in the presence or absence of O(2). While the accumulation of lactate is thought to be a major contributor to the reduction of pH-circumscribing aggressive tumors, it is not known if other endogenous metabolic products contribute this acidity. Furthermore, anaerobic metabolism in cancer cells bears similarity to homo-fermentative lactic acid bacteria, however very little is known about an alternative pathway that may drive adenosine triphosphate (ATP) production independent of glycolysis. In this study, we quantify over 40 end-products (amines, acids, alcohols, aldehydes, or ketones) produced by malignant neuroblastoma under accelerated glycolysis (+glucose (GLU) supply 1-10 mM) +/- mitochondrial toxin; 1-methyl-4-phenylpyridinium (MPP(+)) to abate aerobic respiration to delineate differences between anaerobic vs. aerobic cell required metabolic pathways. The data show that an acceleration of anaerobic glycolysis prompts an expected reduction in extracellular pH (pH(ex)) from neutral to 6.7 +/- 0.006. Diverse metabolic acids associated with this drop in acidity were quantified by ionic exchange liquid chromatography (LC), showing concomitant rise in lactate (Ctrls 7.5 +/- 0.5 mM; +GLU 12.35 +/- 1.3 mM; +GLU + MPP 18.1 +/- 1.8 mM), acetate (Ctrl 0.84 +/- 0.13 mM: +GLU 1.3 +/- 0.15 mM; +GLU + MPP 2.7 +/- 0.4 mM), fumarate, and a-ketoglutarate (<10 microM) while a range of other metabolic organic acids remained undetected. Amino acids quantified by o-phthalaldehyde precolumn derivatization/electrochemical detection-LC show accumulation of L: -alanine (1.6 +/- .052 mM), L: -glutamate (285 +/- 9.7 microM), L: -asparagine (202 +/- 2.1 microM), and L: -aspartate (84.2 +/- 4.9 microM) produced during routine metabolism, while other amino acids remain undetected. In contrast, the data show no evidence for accumulation of acetaldehyde, aldehydes, or ketones (Purpald/2,4-dinitrophenylhydrazine-Brady's reagent), acetoin (Voges-Proskauer test), or alcohols (NAD(+)-linked alcohol dehydrogenase). In conclusion, these results provide preliminary evidence to suggest the existence of an active pyruvate-alanine transaminase or phosphotransacetylase/acetyl-CoA synthetase pathway to be involved with anaerobic energy metabolism of cancer cells.
"In comparison of the human CRYM active site with those of AfAlaDH and PpOCD (Gallagher et al. 2004; Goodman et al. 2004), the thyroid hormone T 3 -binding site is proposed. The fact that the T 3 -binding capacity of human CRYM is regulated by the cofactor NADPH (Vie et al. 1997) suggests that the T 3 -binding site should be close to the NADPH molecule or structural changes should be triggered upon NADPH binding. "
[Show abstract][Hide abstract] ABSTRACT: Human cytosolic 3,5,3'-triiodo-L-thyronine-binding protein, also called mu-crystallin or CRYM, plays important physiological roles in transporting 3,5,3'-triiodo-L-thyronine (T(3)) into nuclei and regulating thyroid-hormone-related gene expression. The crystal structure of human CRYM's bacterial homolog Pseudomonas putida ornithine cyclodeaminase and Archaeoglobus fulgidus alanine dehydrogenase have been available, but no CRYM structure has been reported. Here, we report the crystal structure of human CRYM bound with NADPH refined to 2.6 A, and there is one dimer in the asymmetric unit. The structure contains two domains: a Rossmann fold-like NADPH-binding domain and a dimerization domain. Different conformations of the loop Arg83-His92 have been observed in two monomers of human CRYM in the same asymmetric unit. The peptide bond of Val89-Pro90 is a trans-configuration in one monomer but a cis-configuration in the other. A detailed comparison of the human mu-crystallin structure with its structurally characterized homologs including the overall comparison and superposition of active sites was conducted. Finally, a putative T(3)-binding site in human CRYM is proposed based on comparison with structural homologs.
Protein Science 12/2006; 16(2):329-335. DOI:10.1110/ps.062556907 · 2.85 Impact Factor
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