ABSTRACT
BACKGROUND: Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive inborn error of glycine metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. METHODS: This study describes the first screening of NKH in cataract captive-bred vervet monkeys (Chlorocebus aethiops). Glycine dehydrogenase (GLDC), aminomethyltransferase (AMT) and glycine cleavage system H protein (GCSH) were prioritized. RESULTS: Mutation analysis of the complete coding sequence of GLDC and AMT revealed six novel single-base substitutions, of which three were non-synonymous missense and three were silent nucleotide changes. CONCLUSION: Although deleterious effects of the three amino acid substitutions were not evaluated, one substitution of GLDC gene (S44R) could be disease-causing because of its drastic amino acid change, affecting amino acids conserved in different primate species. This study confirms the diagnosis of NKH for the first time in vervet monkeys with cataracts.
Subject(s)
Aminomethyltransferase/genetics , Cataract/veterinary , Chlorocebus aethiops , Glycine Decarboxylase Complex H-Protein/genetics , Glycine Dehydrogenase/genetics , Hyperglycinemia, Nonketotic/veterinary , Monkey Diseases/genetics , Point Mutation , Amino Acid Sequence , Aminomethyltransferase/chemistry , Aminomethyltransferase/metabolism , Animals , Cataract/genetics , Glycine Decarboxylase Complex H-Protein/chemistry , Glycine Decarboxylase Complex H-Protein/metabolism , Glycine Dehydrogenase/chemistry , Glycine Dehydrogenase/metabolism , Hyperglycinemia, Nonketotic/genetics , Mutation, MissenseABSTRACT
Acinetobacter baylyi, a ubiquitous soil bacterium, can cope with high salinity by uptake of choline as precursor of the compatible solute glycine betaine. Here, we report on the identification of a choline dehydrogenase (BetA) and a glycine betaine aldehyde dehydrogenase (BetB) mediating the oxidation of choline to glycine betaine. The betAB genes were found to form an operon together with the potential transcriptional regulator betI. The transcription of the betIBA operon and the two recently identified choline transporters was upregulated in response to choline and choline plus salt. The finding that the osmo-independent transporter BetT1 undergoes a higher upregulation in response to choline alone than betT2 suggests that BetT1 does not primarily function in osmoadaptation. Electrophoretic mobility shift assays led to the conclusion that BetI mediates transcriptional regulation of both, the betIBA gene operon and the choline transporters. BetI was released from the DNA in response to choline which together with the transcriptional upregulation of the bet genes in the presence of choline suggests that BetI is a choline sensing transcriptional repressor.
Subject(s)
Acinetobacter/physiology , Betaine/metabolism , Biosynthetic Pathways/genetics , Choline/metabolism , Gene Expression Regulation, Bacterial , Osmoregulation , Repressor Proteins/metabolism , Acinetobacter/genetics , Acinetobacter/metabolism , Choline Dehydrogenase/genetics , Choline Dehydrogenase/metabolism , Electrophoretic Mobility Shift Assay , Glycine Dehydrogenase/genetics , Glycine Dehydrogenase/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Operon , Oxidation-Reduction , Transcription, GeneticABSTRACT
The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.
Subject(s)
Alanine Dehydrogenase/genetics , Alanine Dehydrogenase/metabolism , Glycine Dehydrogenase/genetics , Glycine Dehydrogenase/metabolism , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Alanine/metabolism , Alanine Dehydrogenase/isolation & purification , Cell Membrane/chemistry , Cloning, Molecular , Cytosol/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Knockout Techniques , Glycine/metabolism , Glycine Dehydrogenase/isolation & purification , Glyoxylates/metabolism , Mycobacterium tuberculosis/chemistry , Nitrogen/metabolism , Pyruvic Acid/metabolismABSTRACT
Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O(2) transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD(+); (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways.
