Kegg Pathway: beta-Alanine metabolism

Pharmacogenet Genomics. 2008 Aug; 18(8): 665-76
Choudhary D, Jansson I, Sarfarazi M, Schenkman JB

OBJECTIVE: The objective of this study was to examine the biochemical and physical properties of cytochrome P450 1B1 (CYP1B1) mutants, test our hypothesis that primary congenital glaucoma (PCG)-causing mutants have altered metabolic activity, and correlate these to structural changes in the molecule. METHODS: CYP1B1.1 cDNA was mutated to four forms found in individuals with the PCG phenotype, Y81N, E229K, A330F, and R368H. Expression and stability of the mutant hemoproteins and their ability to metabolize beta-estradiol, arachidonic acid, and retinoids, were determined. Alterations in mutant properties were related to structural changes by in silico examination, on the basis of the CYP1A2 crystal structure. RESULTS: CYP1B1 mutations strongly affected the stability, ease of heterologous expression, and enzymatic properties of the protein. These were related to the location of the amino acid substitutions in the CYP1B1 structure. Three of the mutations involve residues located on the surface of CYP1B1, Y81N, and E229K near the distal surface, and R368H near the proximal surface. The former two substitutions, Y81N and E229K, caused greatly reduced stability at 4 degrees C. Y81N severely inhibited all substrate turnover, but E229K only inhibited arachidonate turnover and exhibited minimal effect on efficiency of retinoid metabolism and estradiol metabolism. The R368H mutation is relatively conservative, affecting charge-pairing with the deeper-located D374, but it severely inhibited metabolism of all substrates tested, and, like Y81N, expression of the enzyme is less facile than CYP1B1wt. The A330F mutation replaces a small alanine by a bulky phenylalanine in the enzyme active site and had major impact on substrate binding, turnover, uncoupling, and metabolite pattern. CONCLUSION: Consistent with the hypothesis, these PCG-related mutations cause identifiable structural changes negatively impacting CYP1B1 biochemistry and stability.

Biol Pharm Bull. 2008 Jun; 31(6): 1205-8
Jaroenporn S, Yamamoto T, Itabashi A, Nakamura K, Azumano I, Watanabe G, Taya K

The effects of pantothenic acid-supplementation on the adrenal secretion of corticosterone and progesterone in male rats were investigated using an in vitro cell culture system. Male rats at 21 d of age were given 0.03% pantothenic acid in their drinking water for 9 weeks. After 9 weeks of treatment, the animals were decapitated, and adrenal cells were cultured in the absence or presence of rat adrenocorticotropic hormone (ACTH; 10(-15) to 10(-10) M) and/or ovine prolactin (oPRL; 10(-9) to 10(-7) M) for 4 h. Adrenal cells in pantothenic acid-treated rats exhibited higher basal levels of corticosterone and progesterone than control rats. The response of ACTH and/or PRL on corticosterone and progesterone release was higher in the pantothenic acid-treated rats than in the control rats. In addition, PRL increased the stimulatory effect of ACTH-induced corticosterone secretion in both normal and pantothenic acid-treated rats. These results clearly demonstrated that pantothenic acid supplementation stimulates the ability of adrenal cells in male rats to secrete corticosterone and progesterone. Additionally, these results also showed that pantothenic acid supplementation induced adrenal hyperresponsiveness to ACTH stimulation, and PRL further stimulated adrenal sensitivity to ACTH.

J Mol Biol. 2008 Jun 20; 379(5): 1094-106
Liu Q, Dansereau JT, Puttamadappa SS, Shekhtman A, Derbyshire V, Belfort M

I-TevI is a modular intron-encoded endonuclease, consisting of an N-terminal catalytic domain and a C-terminal DNA-binding domain, joined by a 75 amino acid linker. This linker can be divided into three regions, starting at the N terminus: the deletion-intolerant (DI) region; the deletion-tolerant (DT) region; and a zinc finger, which acts as a distance determinant for cleavage. To further explore linker function, we generated deletion and substitution mutants that were tested for their preference to cleave at a particular distance or at the correct sequence. Our results demonstrate that the I-TevI linker is multi-functional, a property that sets it apart from junction sequences in most other proteins. First, the linker DI region has a role in I-TevI cleavage activity. Second, the DT linker region participates in distance determination, as evident from DT mutants that display a phenotype similar to that of the zinc-finger mutants in their selection of a cleavage site. Finally, NMR analysis of a freestanding 56 residue linker segment showed an unstructured stretch corresponding to the DI region and a portion of the DT region, followed by a beta-strand corresponding to the remainder of the DT region and containing a key distance-determining arginine, R129. Mutation of this arginine to alanine abolished distance determination and disrupted the beta-strand, indicating that the structure of the DT linker region has a role in cleavage at a fixed distance.

Plant J. 2008 May 20;
Weigelt K, Küster H, Radchuk R, Müller M, Weichert H, Fait A, Fernie AR, Saalbach I, Weber H

Nitrogen application to legumes regulates seed metabolism and composition. We recently showed that seed-specific overexpression of amino acid permease VfAAP1, increases amino acid supply, seed N and protein. Two consecutive field trials using Pisum sativum-AAP1-lines confirmed increases in seed N and globulin content, however, compensatory changes of sucrose/starch and individual seed weight were observed. We present a comprehensive analysis of AAP1-seeds using combinatorial transcript and metabolite profiling to monitor effects of nitrogen supply on seed metabolism. AAP1-seeds have increased amino acids and stimulated gene expression associated to storage protein synthesis, maturation, deposition and vesicle trafficking. Transcript/metabolite changes reveal channelling of surplus N into transient storage pools asparagine and arginine, indicating that asparagine-synthase is transcriptionally activated by high N status and/or C limitation. Increased C-acceptor demand for amino acid synthesis due to elevated seed N initiates sucrose mobilisation and sucrose dependent pathways via sucrose synthase, glycolysis and the TCA-cycle. AAP1-seeds display C-limitation, leading to catabolism of arginine, glutamic acid and methionine to putrescine, beta-Alanine and succinate. Mitochondria are involved in the coordination of C/N metabolism with branched-chain amino acid catabolism and gamma-amino-butyric acid-shunt. AAP1-seeds contain higher abscisic acid, possibly involved in storage-associated gene expression and N-dependent stimulation of sucrose mobilisation indicating a signalling network of C, N and ABA operating during seed maturation. These results demonstrate that legume seeds have high capacity to regulate N to C ratios and highlight the importance of mitochondria in the control of N to C balance and amino acid homeostasis.

Exp Diabetes Res. 2008; 2008: 371716
Amartey JK, Shi Y, Al-Jammaz I, Esguerra C, Al-Otaibi B, Al-Mohanna F

An imaging method capable of using a signal from pancreatic beta cells to determine their mass would be of immense value in monitoring the progression of diabetes as well as response to treatment. Somatostatin receptors (SSTRs) are expressed on beta cells and are a potential target for imaging. The main objective of this study was to investigate whether pancreatic beta cells are a target for radiolabeled naphthylalanine derivatives. The molecules were subjected to in vitro and ex vivo evaluations. Pancreatic uptake of radioactivity was lower in nonobese diabetic (NOD) mice than normal mice at all time points investigated (P < .05) and correlated with the number of islets in tissue sections of both control and NOD mice. Immunohistochemical and confocal fluorescent microscopic studies showed colocalization of insulin and the conjugate radioligand in the pancreas. The results demonstrated that pancreatic uptake is receptor-mediated, and that beta cells are the primary target.

J Am Chem Soc. 2008 Jun 11; 130(23): 7172-3
Sackewitz M, Scheidt HA, Lodderstedt G, Schierhorn A, Schwarz E, Huster D

The nuclear poly(A) binding protein PABPN1 possesses a natural 10 alanine stretch that can be extended to 17 Ala by codon expansion. The expansions are associated with the disease oculopharyngeal muscular dystrophy (OPMD), which is characterized histopathologically by intranuclear fibrillar deposits. Here, we have studied the Ala extended fibrillar N-terminal fragment of PABPN1, (N-(+7)Ala), comprising 152 amino acids. At natural abundance, cross-polarized 13C MAS NMR spectra are dominated by the three Ala signals with characteristic beta-sheet chemical shifts. In contrast, directly polarized 13C MAS spectra show a multitude of narrow lines, suggesting a large portion of highly mobile sites. Proteolytic cleavage of the protein combined with MALDI-TOF mass spectrometry revealed a protease-resistant peptide encompassing residues 13/14 to 50-52 with the poly-Ala stretch in the center. Measurements of the 1H-13Calpha dipolar couplings of 13C/15N-labeled N-(+7)Ala revealed high order parameters of 0.77 for the poly-Ala stretch of the fibril, while the majority of the residues of N-(+7)Ala exhibited very low order parameters between 0.06 and 0.15. Only some Gly residues that are flanking the Ala-rich region had significant order parameters of 0.47. Thus, site-specific dynamic mapping represents a useful tool to identify the topology of fibrillar proteins.

Pediatr Crit Care Med. 2008 Mar; 9(2): 209-16
Jeschke MG, Finnerty CC, Kulp GA, Przkora R, Mlcak RP, Herndon DN

OBJECTIVE: Recombinant human growth hormone (rhGH) is a salutary modulator of posttraumatic metabolic responses. However, rhGH administration is associated with deleterious side effects, such as hyperglycemia, increased free fatty acids, and triglycerides, which limit its use. Administration of beta-blocker attenuates cardiac work and resting energy expenditure after severe thermal injury and improves fat metabolism and insulin sensitivity. Therefore, the combination of rhGH plus propranolol appears ideal. The aim of the present study was to determine whether rhGH plus propranolol improves hypermetabolism and the inflammatory and acute phase response after severe burn without causing adverse side effects. DESIGN: Prospective randomized control trial. SETTING: Shriners Hospitals for Children. PATIENTS: Fifteen pediatric patients with burns > 40% total body surface area, 0.1-16 yrs of age, admitted within 7 days after burn. Fifteen children were matched for burn size, age, gender, inhalation injury, and infection and served as controls. INTERVENTIONS: Patients in the experimental group received rhGH (0.2 mg/kg/day) and propranolol (to decrease heart rate by 15%) for > or = 15 days. MEASUREMENTS AND MAIN RESULTS: Outcome measurements included resting energy expenditure, body composition, acute phase proteins, and cytokines. Both cohorts were similar in age, burn size, gender, and accompanying injuries. Percent predicted resting energy expenditure significantly decreased in patients receiving rhGH/propranolol (Delta -5% +/- 8%) compared with controls (Delta +35% +/- 20%) (p < .05). rhGH/propranolol administration significantly decreased serum C-reactive protein, cortisone, aspartate aminotransferase, alanine aminotransferase, free fatty acids, interleukin-6, interleukin-8, and macrophage inflammatory protein-1beta when compared with controls, while growth hormone/propranolol increased serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, growth hormone, prealbumin, and interleukin-7 when compared with placebo (p < .05). CONCLUSIONS: rhGH in combination with propranolol attenuates hypermetabolism and inflammation without the adverse side effects found with rhGH therapy alone.

Drug Metab Dispos. 2008 May 12;
Cooper AJ, Younis IR, Niatsetskaya ZV, Krasnikov BF, Pinto JT, Petros WP, Callery PS

The present work documents the first example of an enzyme-catalyzed beta-elimination of a thioether from a sulfonium cysteine S-conjugate. beta-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a non-enzymatic beta-elimination reaction at pH 7.4 and 37 degrees C to yield tetrahydrothiophene, pyruvate and ammonia. This reaction is accelerated by a) rat liver, kidney and brain homogenates, b) isolated rat liver mitochondria, and c) pyridoxal 5'-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a beta-lyase reaction with THT-A was identified as cystathionine gamma-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

Bioorg Med Chem Lett. 2008 Jun 1; 18(11): 3206-10
Masuda Y, Uemura S, Nakanishi A, Ohashi R, Takegoshi K, Shimizu T, Shirasawa T, Irie K

Structural analysis of 42-residue amyloid beta (Abeta42) aggregates using rotational resonance in solid-state NMR verified that C(beta) and/or C(gamma) of Met-35 and the carboxyl carbon of Ala-42 are proximal enough to form an intramolecular antiparallel beta-sheet in the C-terminus. The S-oxidized radical cation at Met-35, an ultimate radical species responsible for neurotoxicity, could be stabilized by the carboxylate anion at the C-terminus, resulting in aggregation to cause long-term oxidative stress.

PLoS Pathog. 2008 Feb 15; 4(2): e1000001
Hett EC, Chao MC, Deng LL, Rubin EJ

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein-protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall-targeting beta-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein-protein interaction as one way of regulating its activity.

J Biol Chem. 2008 Jul 4; 283(27): 18765-72
Christoffersen C, Ahnström J, Axler O, Christensen EI, Dahlbäck B, Nielsen LB

Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH(2)-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoM(Q22A) cDNA in the liver (apoM(Q22A)-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoM(Q22A) results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoM(Q22A)-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoM(Q22A)-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoM(Q22A)-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney.

Biochemistry. 2008 May 27; 47(21): 5724-35
Brown G, Singer A, Proudfoot M, Skarina T, Kim Y, Chang C, Dementieva I, Kuznetsova E, Gonzalez CF, Joachimiak A, Savchenko A, Yakunin AF

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 May 1; 64(Pt 5): 432-4
Ambrazi M, Fellas G, Kapetaniou EG, Kotsifaki D, Providaki M, Kokkinidis M

Rop is the paradigm of a canonical four-alpha-helical bundle. Its loop region has attracted considerable interest because a single alanine-to-proline substitution (A31P) in the loop is sufficient to change the topology of this small protein. In order to further analyse the loop region as a possible folding-control element, the double mutant D30P/A31G (RopPG) was produced, purified and crystallized. The crystals belonged to space group P2(1), with unit-cell parameters a = 26.7, b = 38.8, c = 56.6 A, beta = 100.9 degrees and two molecules in the asymmetric unit. A complete data set was collected at 100 K to a resolution of 1.4 A using synchrotron radiation.

J Mol Biol. 2008 May 30; 379(2): 243-50
Andersen B, Lundgren S, Dobritzsch D, Piskur J

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-Alanine to beta-Alanine, is catalyzed by two beta-Alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.

J Biol Chem. 2008 Jun 20; 283(25): 17238-49
Li C, Nissim I, Chen P, Buettger C, Najafi H, Daikhin Y, Nissim I, Collins HW, Yudkoff M, Stanley CA, Matschinsky FM

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.

Tohoku J Exp Med. 2008 Apr; 214(4): 321-5
Kotani K, Shimohiro H, Adachi S, Sakane N

Gamma-glutamyl transferase (GGT) is an enzyme present in serum and on most cell surfaces and serves as an oxidative stress marker. Although serum GGT is associated with hypertension development, little data are available on the associations between GGT and hypertension among populations with diabetes mellitus (DM). Our aim was to investigate the potential association between the changes in systolic or diastolic blood pressure (SBP/DBP) and the GGT level in type 2 DM subjects, in comparison with non-DM subjects. In 179 non-DM and 177 DM subjects, SBP/DBP, body mass index (BMI), fasting plasma glucose, serum asparate aminotransferase, alanine aminotransferase and GGT were measured at the baseline and after a 1-year period. Between these 2-measurement points, in non-DM subjects, SBP and DBP levels were significantly increased, while GGT tended to increase. In contrast, in DM subjects, the mean levels of SBP, DBP and GGT remained unchanged. Multivariate analysis revealed that in non-DM subjects the degree of increase in SBP was significantly and positively correlated to that of GGT (beta = 0.165), along with age and BMI. Likewise, the increase in DBP was correlated to that of GGT in non-DM subjects (beta = 0.170). In contrast, in DM subjects, the degree of increase in SBP was significantly correlated to that of only GGT (beta = 0.166). These results suggest that the presence of DM may attenuate the effects of GGT on DBP.

FEBS Lett. 2008 May 28; 582(12): 1661-6
Atherton HJ, Jones OA, Malik S, Miska EA, Griffin JL

Proton Nuclear Magnetic Resonance spectroscopy and Gas Chromatography Mass Spectrometry based metabolomics has been used in conjunction with multivariate statistics to examine the metabolic changes in Caenorhabditis elegans following the deletion of nuclear hormone receptor-49 (nhr-49). Deletion of the receptor produced profound changes in fatty acid metabolism, in particular an increase in the ratio of unsaturated to saturated fatty acids, a decrease in the concentration of glucose and increases in lactate and alanine. Given the proposed functional similarity between nhr-49 and the mammalian peroxisome proliferator-activated receptors (PPARs) these changes were compared with the metabolome of the PPAR-alpha null mouse. The metabolomic approach demonstrated a number of similarities including the regulation of lipid synthesis, beta-oxidation of fatty acids and changes in glycolysis/gluconeogenesis.

Appl Environ Microbiol. 2008 Jun; 74(11): 3434-43
Sallam A, Steinbüchel A

Four bacterial strains were isolated from a cyanophycin granule polypeptide (CGP)-degrading anaerobic consortium, identified by 16S rRNA gene sequencing, and assigned to species of the genera Pseudomonas, Enterococcus, Clostridium, and Paenibacillus. The consortium member responsible for CGP degradation was assigned as Pseudomonas alcaligenes strain DIP1. The growth of and CGP degradation by strain DIP1 under anaerobic conditions were enhanced but not dependent on the presence of nitrate as an electron acceptor. CGP was hydrolyzed to its constituting beta-Asp-Arg dipeptides, which were then completely utilized within 25 and 4 days under anaerobic and aerobic conditions, respectively. The end products of CGP degradation by strain DIP1 were alanine, succinate, and ornithine as determined by high-performance liquid chromatography analysis. The facultative anaerobic Enterococcus casseliflavus strain ELS3 and the strictly anaerobic Clostridium sulfidogenes strain SGB2 were coisolates and utilized the beta-linked isodipeptides from the common pool available to the mixed consortium, while the fourth isolate, Paenibacillus odorifer strain PNF4, did not play a direct role in the biodegradation of CGP. Several syntrophic interactions affecting CGP degradation, such as substrate utilization, the reduction of electron acceptors, and aeration, were elucidated. This study demonstrates the first investigation of CGP degradation under both anaerobic and aerobic conditions by one bacterial strain, with regard to the physiological role of other bacteria in a mixed consortium.

FEBS J. 2008 Jun; 275(11): 2754-64
Ronconi S, Jonczyk R, Genschel U

The linear biosynthetic pathway leading from alpha-ketoisovalerate to pantothenate (vitamin B5) and on to CoA comprises eight steps in the Bacteria and Eukaryota. Genes for up to six steps of this pathway can be identified by sequence homology in individual archaeal genomes. However, there are no archaeal homologs to known isoforms of pantothenate synthetase (PS) or pantothenate kinase. Using comparative genomics, we previously identified two conserved archaeal protein families as the best candidates for the missing steps. Here we report the characterization of the predicted PS gene from Methanosarcina mazei, which encodes a hypothetical protein (MM2281) with no obvious homologs outside its own family. When expressed in Escherichia coli, MM2281 partially complemented an auxotrophic mutant without PS activity. Purified recombinant MM2281 showed no PS activity on its own, but the enzyme enabled substantial synthesis of [14C]4'-phosphopantothenate from [14C]beta-Alanine, pantoate and ATP when coupled with E. coli pantothenate kinase. ADP, but not AMP, was detected as a coproduct of the coupled reaction. MM2281 also transferred the 14C-label from [14C]beta-Alanine to pantothenate in the presence of pantoate and ADP, presumably through isotope exchange. No exchange took place when pantoate was removed or ADP replaced with AMP. Our results indicate that MM2281 represents a novel type of PS that forms ADP and is strongly inhibited by its product pantothenate. These properties differ substantially from those of bacterial PS, and may explain why PS genes, in contrast to other pantothenate biosynthetic genes, were not exchanged horizontally between the Bacteria and Archaea.

J Biochem Mol Toxicol. 2008; 22(2): 119-27
Tripathi S, Somashekar BS, Mahdi AA, Gupta A, Mahdi F, Hasan M, Roy R, Khetrapal CL

The toxic effects of Al(3+) have been studied in 90-days AlCl(3) orally treated male albino rats (n = 7) using (1)H NMR spectroscopy-based metabolic profile of rat serum and urine, serum enzyme tests, behavioral impairment, and histopathology of kidney and liver. Metabolic profile of 90-days Al(3+)-treated rat sera showed significantly elevated levels of alanine, glutamine, beta-hydroxy-butyrate, and acetoacetate and significantly decreased level of acetone when compared with that of control rats. However, metabolic profile of 90-days Al(3+)-treated rat urine showed significantly decreased levels of citrate, creatinine, allantoin, trans-aconitate, and succinate and significantly increased level of acetate when compared to control rats. The overall perturbations observed in the metabolic profile of serum and urine demonstrate the impairment in the tricarboxylic acid cycle, liver and kidney metabolism, which was further reinstated by clinical chemistry and histopathological observations. Moreover, "in vivo" behavioral impairment has also been observed as the indication of aluminum neurotoxicity.