Utilization Of Alanine Research Articles

Abstract #1184551: Hypertriglyceridemic pancreatitis in a rare case of type A pyruvate carboxylase deficiency

Pyruvate carboxylase (PC) deficiency is a spectrum of rare autosomal recessive disorders that can have significant metabolic consequences. Decreases of PC activity limits utilization of lactate and alanine for anaplerotic and synthetic fluxes. PC deficiency can lead to hypoglycemia, lactic acidosis, and hyperammonemia. Patients can develop central nervous system disease (via glial damage and demyelination leading to hydrocephalus), hepatotoxicity and insulinopenia. The latter can result in hypertriglyceridemia, though not a hallmark of PC deficiency.

Genomic differences among strains of Corynebacterium cystitidis isolated from uterus of camels.

Members of the Corynebacterium cystitidis species are usually isolated from kidney and urine of cow having pyelonephritis. Nevertheless, we have isolated Corynebacterium cystitidis for the first time from uterus of camels, extending the type of mammalian host for this species. Furthermore, it remains unknown whether there are significant genetic variations between strains isolated from different host species and anatomic sites. In this perspective, we investigated the genomic diversity of Corynebacterium cystitidis species, whose pan genome remain unexplored to date. Thus, we sequenced and compared the genomes of five Corynebacterium cystitidis of camel origin and a public genome of cow associated Corynebacterium cystitidis. Results revealed open pan genome of 4,038 gene clusters and horizontal gene transfer played a role in the extensive genetic diversity. Further, we found an obvious distinction between cow and camel associated C. cystitidis via phylogenomic analysis and by average nucleotide identity value of 95% between the two distant lineages and > 99% within camel associated C. cystitidis strains. Moreover, our data supports the hypothesis that the gene repertoire of cow associated Corynebacterium cystitidis developed so as to become more adaptable to the urine milieu. These genetic potentials are specifically evident for genes required for benzoate breakdown, iron transport, citrate and alanine utilization. Our findings confirm the differentiation of strains into camel lineage and cow lineage. These different niches, comprising the uterus of camel and urinary tract of cow probably played a role in shaping the gene repertoire of strains.

Alanine Catabolism of Paneth Cells Maintains Intestinal Stem Cell Function During Dietary Restriction

Paneth cell niche promotes the function of intestinal stem cells (ISCs) during reduced food intake, but how ISC activity can be boosted when availability of resources is simultaneously reduced remains unknown. Here, we show that the increase in ISC function upon dietary restriction (DR) is dependent on the mitochondrial alanine transaminase Gpt2 in Paneth cells. Metabolic tracing of Alanine directly showed that during DR, the Paneth cell niche catabolizes increased amounts of alanine to produce lactate, which is transported to ISCs to fuel their TCA cycle. Correspondingly, inhibition of lactate import to stem cells abolished the DR induced capacity of Paneth cells to support ISC function. DR resulted in deacetylation of Gpt2 in a Sirtuin3 dependent manner, providing a possible mechanism for Gpt2 regulation. Taken together, our results reveal an important role of the metabolic niche in tissue adaptation, where alanine utilization by the Paneth cells supports stem cells during nutritional challenges.

Chemically unidentified dissolved organic carbon: A pivotal piece for microbial activity in a productive area of the Northern Patagonian shelf

The biochemical composition and fluorescence properties of DOM were assessed in relation to phytoplankton and major aquatic bacterial clades in a regenerative area of the Argentine Shelf. DOM was mainly of autochthonous biological origin, containing humic- and protein-like substances of medium degree of unsaturation and diagenesis. Biochemical-DOM accounted for 25% of total DOC, being dissolved combined amino acids (DCAA) the dominant fraction followed by free carbohydrates. Phytoplankton was the main source of serine, alanine, and valine, and particulate carbohydrates. Gammaproteobacteria abundance correlated negatively with ammonium and positively with DCAA, suggesting a coupling between ammonium consumption and refractory amino acid production. A preferential utilization of alanine, leucine and threonine as nitrogen source was inferred from the distribution of Cytophaga-Flavobacteria-Bacteroidete in relation with dissolved free amino acids (DFAA). Notably, Alpha- and Betaproteobacteria correlated with the large pool (75%) of chemically unidentified DOC and not with DCAA or dissolved combined carbohydrates. Particularly, Alphaproteobacteria (∼40% of EUB total heterotrophic bacteria) either significantly contribute to the production of the “humic”, refractory fraction of marine DOM, or the latter impairs resource control on their abundance. Spatial heterogeneity inherent to coastal-shelf areas drives important regional variability in the biochemical properties of DOM.

Mitochondrial metabolic substrate utilization in granulosa cells reflects body mass index and total follicle stimulating hormone dosage in in vitro fertilization patients

To utilize a novel mitochondrial function assay with pooled granulosa cells to determine whether mitochondrial function would differ by patient demographics and embryo development. This was a prospective pilot study in a hospital-based assisted reproductive program and public university. Mitochondrial metabolic substrate utilization was assessed in pooled granulosa cells from 40 women undergoing in vitro fertilization during 2018 and 2019. Assessment of mitochondrial substrate metabolism in pooled granulosa cells revealed higher citric acid, L-malic acid, and octanoyl-L-carnitine utilization with higher body mass index (BMI). Utilization of citric acid, cis-aconitic acid, D-alpha-keto-glutaric acid, L-glutamine, and alanine plus glycine was significantly lower as total dosage of FSH administered increased. Utilization of glycogen was significantly higher in patients with a higher percentage of fertilized oocytes. D-alpha-keto-glutaric acid utilization was significantly lower in patients with a higher percentage of good 8-cell embryos. L-glutamine utilization was significantly lower, with a higher percentage of blastocyst formation. Mitochondrial metabolic scores (MMS), which reflect overall mitochondrial activity of the granulosa pool, were significantly higher in patients with higher BMI and with greater numbers of mature oocytes retrieved. MMS in granulosa decreased as total FSH dose administered increased. Granulosa cell utilization of substrates feeding into the citric acid cycle changed with total FSH dosage and BMI. Fertilization rate, 8-cell embryo quality, and blastocyst formation also associated with different energy substrate usage. Mitochondrial substrate utilization by granulosa cells from individual follicles could be further developed into a useful diagnostic tool.

Untargeted Lipidomics Highlight the Depletion of Deoxyceramides during Therapy-Induced Senescence.

Therapy-induced senescence is a state of cell cycle arrest that occurs as a response to various chemotherapeutic reagents, especially ones that cause DNA damage. Senescent cells display resistance to cell death and can impair the efficacy of chemotherapeutic strategies. Since lipids can exhibit pro-survival activity, it is envisioned in this article that probing the lipidome could provide insights into novel lipids that are involved in senescence. Therefore, a tissue culture model system is established and the cellular lipidomes of senescent and proliferating cells are comparatively analyzed. Out of thousands of features detected, 17 species are identified that show significant changes in senescent cells. The majority of these species (11 out of 17) are atypical sphingolipids, 1-deoxyceramides/dihydroceramides, which are produced as a result of the utilization of alanine, instead of serine during sphingolipid biosynthesis. These lipids are depleted in senescent cells. Elevating the levels of deoxyceramides by supplementing the growth medium with metabolic precursors or by directly adding deoxyceramide result in decreased senescence, suggesting that these species might play a key role in this process.

Characterization and survey in cattle of a rumen Pyrimadobacter sp. which degrades the plant toxin fluoroacetate.

Among the natural halogenic compounds, the plant toxin fluoroacetate (FA) causes livestock fatalities in southern hemisphere countries. Here, we report on the isolation of a rumen bacterium, strain C12-8 that degrades FA under anaerobic conditions. 16S rRNA gene sequence analysis showed this bacterium belonged to the Pyramidobacter genus within the Synergistetes phylum and was 98% similar to Pyramidobacter piscolens W5455 isolated from the human oral cavity. Transmission electron microscopy showed the cell envelope to be unusual, with only one membrane and no obvious external wall. Growth and FA degradation were enhanced by peptide-rich protein hydrolysates but not carbohydrates. End products of metabolism were mainly acetate, propionate/isovalerate and isobutyrate. Strain C12-8 preferentially used peptide-bound amino acids rather than free amino acids. Glycine, serine, threonine, leucine, histidine and isoleucine were utilized as free and peptide-bound amino acids, but there was minimal utilization of alanine, proline, methionine, aspartic acid, lysine and arginine in either form. A survey of several cattle properties in northern Australia showed that strain C12-8 and other FA degrading bacteria affiliated with Cloacibacillus porcorum strain MFA1 were endemic to cattle in the northern beef herd and may help to reduce toxicity.

Alanine dehydrogenases in mycobacteria.

Since NAD(H)-dependent L-alanine dehydrogenase (EC 1.1.4.1; Ald) was identified as one of the major antigens present in culture filtrates of Mycobacterium tuberculosis, many studies on the enzyme have been conducted. Ald catalyzes the reversible conversion of pyruvate to alanine with concomitant oxidation of NADH to NAD+ and has a homohexameric quaternary structure. Expression of the ald genes was observed to be strongly upregulated in M. tuberculosis and Mycobacterium smegmatis grown in the presence of alanine. Furthermore, expression of the ald genes in some mycobacteria was observed to increase under respiration-inhibitory conditions such as oxygen-limiting and nutrient-starvation conditions. Upregulation of ald expression by alanine or under respiration-inhibitory conditions is mediated by AldR, a member of the Lrp/AsnC family of transcriptional regulators. Mycobacterial Alds were demonstrated to be the enzymes required for utilization of alanine as a nitrogen source and to help mycobacteria survive under respiration-inhibitory conditions by maintaining cellular NADH/NAD+ homeostasis. Several inhibitors of Ald have been developed, and their application in combination with respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline was recently suggested.

Tsc3 regulates SPT amino acid choice in Saccharomyces cerevisiae by promoting alanine in the sphingolipid pathway

The generation of most sphingolipids (SPLs) starts with condensation between serine and an activated long-chain fatty acid catalyzed by serine palmitoyltransferase (SPT). SPT can also use other amino acids to generate small quantities of noncanonical SPLs. The balance between serine-derived and noncanonical SPLs is pivotal; for example, hereditary sensory and autonomic neuropathy type I results from SPT mutations that cause an abnormal accumulation of alanine-derived SPLs. The regulatory mechanism for SPT amino acid selectivity and physiological functions of noncanonical SPLs are unknown. We investigated SPT selection of amino acid substrates by measuring condensation products of serine and alanine in yeast cultures and SPT use of serine and alanine in a TSC3 knockout model. We identified the Tsc3 subunit of SPT as a regulator of amino acid substrate selectivity by demonstrating its primary function in promoting alanine utilization by SPT and confirmed its requirement for the inhibitory effect of alanine on SPT utilization of serine. Moreover, we observed downstream metabolic consequences to Tsc3 loss: serine influx into the SPL biosynthesis pathway increased through Ypk1-depenedent activation of SPT and ceramide synthases. This Ypk1-dependent activation of serine influx after Tsc3 knockout suggests a potential function for deoxy-sphingoid bases in modulating Ypk1 signaling.

Stage-specific expression of the proline-alanine transporter in the human pathogen Leishmania

Leishmania are obligatory intracellular parasites that cycle between the sand fly midgut (extracellular promastigotes) and mammalian macrophage phagolysosomes (intracellular amastigotes). They have developed mechanisms of adaptation to the distinct environments of host and vector that favor utilization of both proline and alanine. LdAAP24 is the L. donovani proline-alanine transporter. It is a member of Leishmania system A that translocates neutral amino acids. Since system A is promastigote-specific, we aimed to assess whether LdAAP24 is also expressed exclusively in promastigotes. Herein, we established that upon exposing L. donovani promastigotes to amastigote differentiation signal (pH 5.5 and 37 °C), parasites rapidly and completely degrade LdAAP24 protein in both axenic and in spleen-derived amastigotes. In contrast, LdAAP24 mRNA remained unchanged throughout differentiation. Addition of either MG132 or Bafilomycin A1 partially inhibited LdAAP24 protein degradation, indicating a role for both lysosome- and proteasome-mediated degradation. This work provides the first evidence for post-translational regulation of stage-specific expression of LdAAP24.

Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique.

Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

Sorption of Alanine changes microbial metabolism in addition to availability

Sorption is one of the main processes stabilizing organic matter in soil against microbial mineralization. We hypothesize that besides reduced accessibility for microorganisms and enzymes, changes in microbial metabolism additionally intensify this organic matter stabilization effect of sorption.Position-specifically 14C labeled Alanine was applied to soil as solution or sorbed on sterilized soil to investigate the mechanisms underlying this metabolism related stabilization effect. Sorption decreased initial mineralization of Alanine by ~80% and doubled the duration until the mineralization maxima (14CO2 peak). Almost all Alanine was taken up by microorganisms independent on sorption, and C‐1 was completely (>99%) decarboxylated during glycolysis after one day. Sorption could not prevent microbial utilization of Alanine, but increased the carbon use efficiency (CUE) of sorbed Alanine for 60% compared to Alanine in solution and increased C incorporation in microbial biomass up to four times. The position-specific pattern of 14C in soil and in microbial biomass showed that oxidation of C-2 from sorbed Alanine was strongly lowered compared to free Alanine. Both higher CUE and delayed C-2 mineralization were achieved by a higher C flux towards efficient anabolism, or/and to slower cycling cell components.Limitation of accessibility for microorganisms alone does not explain the stabilizing effect of sorption on organic substances like amino acids and the observed changed position specific pattern. Even though all sorbed Alanine was taken up by microorganisms within 3days, C partitioning towards anabolism, slower microbial turnover and increased CUE increased C retention from sorbed compounds in soil even after microbial uptake. Position-specific labeling clearly showed that LMWOS are stabilized by sorption not as intact molecules, but after microbial metabolization – as released metabolites or microbial biomass. We conclude that the indirect effects of sorption, namely 1) more C partitioned to anabolism, 2) slower decomposition, 3) higher incorporation into microbial biomass and 4) increased carbon use efficiency promote C retention in soil and may be even more important than the direct effect, namely inaccessibility. The finding that stabilization did not significantly impede microbial utilization, but sorption greatly increased carbon use efficiency has major implications for conceptual and numerical representation of organic matter stabilization and losses in soils.

Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series.

Glucose and oxygen are two of the most important molecules transferred from mother to fetus during eutherian pregnancy, and the metabolic fates of these nutrients converge at the transport and metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex in the inner mitochondrial membrane that consists of two essential components, MPC1 and MPC2. Here, we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. Mpc1 deletion was homozygous lethal in midgestation, but Mpc1 hypomorphs and tissue-specific deletion of Mpc1 presented as early perinatal lethality. The allelic series demonstrated that graded suppression of MPC resulted in dose-dependent metabolic and transcriptional changes. Steady-state metabolomics analysis of brain and liver from Mpc1 hypomorphic embryos identified compensatory changes in amino acid and lipid metabolism. Flux assays in Mpc1-deficient embryonic fibroblasts also reflected these changes, including a dramatic increase in mitochondrial alanine utilization. The mitochondrial alanine transaminase GPT2 was found to be necessary and sufficient for increased alanine flux upon MPC inhibition. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be mitigated in part by alternative anaplerotic substrates in utero.

Dynamics of amino acid utilization in Phaeobacter inhibensDSM 17395

Time-resolved utilization of multiple amino acids by Phaeobacter inhibens DSM 17395 was studied during growth with casamino acids. The 15 detected amino acids could be grouped according to depletion rate into four different categories, i.e. from rapid (category I) to nondepletion (category IV). Upon entry into stationary growth phase, amino acids of category I (e.g. glutamate) were (almost) completely depleted, while those of categories II (e.g. leucine) and III (e.g. serine) were further consumed at varying rates and to different extents. Thus, cultures entered stationary growth phase despite the ample presence of organic nutrients, i.e. under nonlimiting conditions. Integrated proteomic and metabolomic analysis identified 1747 proteins and 94 intracellular metabolites. Of these, 180 proteins and 86 metabolites displayed altered abundance levels during growth. Most strikingly, abundance and activity profiles of alanine dehydrogenase concomitantly increased with the onset of enhanced alanine utilization during transition into stationary growth phase. Most enzymes of amino acid and central metabolism, however, displayed unaltered abundances across exponential and stationary growth phases. In contrast, metabolites of the Entner-Doudoroff pathway and gluconeogenesis as well as cellular fatty acids increased markedly in abundance in early stationary growth phase.

해양 홍조류 Laurencia sp. (Ceramiales: Rhodomelaceae)에서 분리한 Oceanisphaera sp. JJM57의 분리 및 동정

A taxonomic study was carried out to assess the phylogenetic characteristics of isolate JJM57 from marine red algae Laurencia sp. collected from intertidal zone in Jeju Island, South Korea. Comparative analysis of 16S rRNA gene sequence shows that this isolate belongs to the genus Oceanisphaera. It shows 98.02% and 97.7% sequence similarity with Oceanisphera litoralis DSM 15406 T and Oceanisphera donghaensis KCTC 12522 T , respectively. Strain JJM57 is a Gram-negative, aerobic, moderately halophilic bacterium able to grow in different NaCl concentration ranges from 0.5 to 8.0% and at varying temperatures from 4 to 37℃. Sharing some of the physiological and biochemical properties with O. litoralis and O. donghaensis, JJM57 strain differs in the utilization of ethanol, proline, and alanine. The G+C contents of the strain JJM57 is 61.94 mol% and it is rich in C 16:1 ω7c and/or iso-C 15:0 2 -OH, C 16:0 , and C 18:1 ω7c fatty acids. The DNA-DNA relatedness data separates the strain JJM57 from other species such as O. litoralis and O. donghaensis. On the basis of these polyphasic evidences, present study proposed that strain JJM57 (=KCTC 22371 =AM983543 =CCUG 60764) represents a novel bacterial species of Oceanisphaera.

Continuous Glucose Monitoring: A Valuable Monitoring Tool for Management of Hypoglycemia During Chemotherapy for Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) maintenance therapy (MT) has been occasionally associated with symptomatic hypoglycemia (SH), attributed to purine analog (mercaptopurine [6-MP]). This hypoglycemia has been hypothesized to affect substrate utilization of gluconeogenic precursor alanine in the liver. An overweight 5-year-old boy with ALL was evaluated for SH (lethargy and vomiting) that occurred 8-10 h after fasting while receiving daily 6-MP. Hypoglycemic episodes (>20 episodes per month) occurred predominantly around midmorning but not during the 5-day dexamethasone pulse. The adrenocorticotropic hormone test yielded a normal cortisol response, which ruled out pituitary adrenal suppression. A 12-h overnight fasting glucose was 49 mg/dL, with suppressed insulin response <2 IU/mL, low C-peptide of 0.5 ng/mL, high insulin-like growth factor-binding protein >160 ng/mL, high free fatty acid of 2.64 mmol/L, and negative glucagon stimulation test (change in blood glucose [BG] <5 mg/dL). These results ruled out hyperinsulinism. The patient was placed on cornstarch therapy 5 h prior to dosing with 6-MP. This treatment reduced the SH events to fewer than two episodes per month. To study the efficacy of cornstarch, the patient was fitted with the iPro™ professional continuous glucose monitoring system (CGMS) (Medtronic MiniMed, Northridge, CA) with a preset low alarm at 70 mg/dL, which was worn for a period of 5 days while the patient was on cornstarch. With 1,000 sensor reading the BG range was 65-158 mg/dL, and the percentage mean absolute difference between sensor and finger-stick BG readings (the parent monitored his BG four times a day) was 9.4%. There were no hypoglycemic episodes detected by the CGMS while the patient was on cornstarch. After the cessation of chemotherapy, a 15-h fasting study was performed, and the CGMS was placed. Results showed resolution of hypoglycemia. The CGMS helped us devise an effective management plan for our patient. CGMS proved useful as an adjunct to characterize the pattern of hypoglycemia and to validate the benefit of cornstarch in hypoglycemia associated with 6-MP treatment of ALL.

Bacterial Responses to a Simulated Colon Tumor Microenvironment

One of the few bacteria that have been consistently linked to colorectal cancer (CRC) is the opportunistic pathogen Streptococcus gallolyticus. Infections with this bacterium are generally regarded as an indicator for colonic malignancy, while the carriage rate of this bacterium in the healthy large intestine is relatively low. We speculated that the physiological changes accompanying the development of CRC might favor the colonization of this bacterium. To investigate whether colon tumor cells can support the survival of S. gallolyticus, this bacterium was grown in spent medium of malignant colonocytes to simulate the altered metabolic conditions in the CRC microenvironment. These in vitro simulations indicated that S. gallolyticus had a significant growth advantage in these spent media, which was not observed for other intestinal bacteria. Under these conditions, bacterial responses were profiled by proteome analysis and metabolic shifts were analyzed by (1)H-NMR-spectroscopy. In silico pathway analysis of the differentially expressed proteins and metabolite analysis indicated that this advantage resulted from the increased utilization of glucose, glucose derivates, and alanine. Together, these data suggest that tumor cell metabolites facilitate the survival of S. gallolyticus, favoring its local outgrowth and providing a possible explanation for the specific association of S. gallolyticus with colonic malignancy.

Regulatory role for l-arginine in the utilization of amino acids by pig small-intestinal bacteria

We recently reported that bacteria from the pig small intestine rapidly utilize and metabolize amino acids (AA). This study investigated the effect of L-arginine on the utilization of AA by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Bacteria were incubated at 37°C for 3 h in anaerobic AA media containing 0-5 mmol/L of arginine to determine the effect of arginine on the bacterial utilization of AA. Amino acids in the medium plus cell extracts were analyzed by high-performance liquid chromatography. Results indicated concentration-dependent increases in the bacterial utilization of arginine and altered fluxes of arginine into ornithine and citrulline in the bacteria. Net glutamine utilization increased in pure bacterial strains with increased concentrations of arginine. With the addition of arginine, net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P<0.05) in Streptococcus sp. and Klebsiella sp., but decreased in E. coli. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by jejunal or ileal mixed bacteria decreased (P<0.05) with the addition of arginine. Complete utilization of asparagine, aspartate and serine were observed in pig small-intestinal bacteria after 3 h of incubation. Overall, the addition of arginine affected the metabolism of the arginine-family of AA and the serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in ileal mixed bacteria. These novel findings indicate that arginine exerts its beneficial effects on swine nutrition partially by regulating AA utilization and metabolism in the small-intestinal microbiota.

New subunits of SPT: New insights into LCB diversity and human disease

Mammalian SPT was originally believed to be a heterodimer of LCB1/SPTLC1 and LCB2a/SPTLC2, but identification of a second LCB2 isoform (Lcb2b/SPTLC3) and the discovery of two small activating subunits, ssSPTa and ssSPTb, has made clear that there are several SPT isozymes. Expression of the human subunits in yeast revealed that isozymes containing ssSPTa have a preference for C16-CoA while those with ssSPTb prefer C18-CoA. Remarkably, this preference has been mapped to a single amino acid, which when changed to the corresponding residue of the other isoform flips acyl-CoA preference. Based on deletion and topology studies, this residue lies in a short cytosolic tail N-terminal to a single transmembrane domain (TMD) within a 35-amino acid core sufficient for full activation. Many of the conserved residues of the ssSPTs reside within the predicted TMD suggesting that interactions between the ssSPTs and the heterodimer are within the membrane. Discovery of the ssSPTs has also provided insight into HSAN1, the most common hereditary sensory neuropathy, which results from mutations in the catalytic subunits of SPT. Originally believed to be catalytically inactive, when expressed with the ssSPTs, the mutant heterotrimers have significant activity and the mutations perturb the active site to allow utilization of alanine as a substrate resulting in the synthesis of deoxy-LCBs that apparently underlie the HSAN1 phenotype.

ALT1-encoded alanine aminotransferase plays a central role in the metabolism of alanine inSaccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the paralogous genes ALT1 and ALT2 have been proposed to encode alanine aminotransferase isozymes. Although in other microorganisms this enzyme constitutes the main pathway for alanine biosynthesis, its role in S. cerevisiae had remained unclear. Results presented in this paper show that under respiratory conditions, Alt1p constitutes the sole pathway for alanine biosynthesis and catabolism, constituting the first example of an alanine aminotransferase that simultaneously carries out both functions. Conversely, under fermentative conditions, it plays a catabolic role and alanine is mainly synthesized through an alternative pathway. It can thus be concluded that ALT1 has functions in alanine biosynthesis and utilization or only alanine utilization under respiratory and fermentative conditions, respectively. ALT2 expression was repressed under all tested conditions, suggesting that Alt2p biosynthesis is strictly controlled and only allowed to express under peculiar physiological conditions.