Mutation analysis of GLDC, AMT and GCSH in cataract captive-bred vervet monkeys (Chlorocebus aethiops).

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.

Glycine decarboxylase is an unusual amino acid decarboxylase involved in tumorigenesis.

Glycine decarboxylase (GLDC) is a metabolic oncogene that links glycine metabolism with tumorigenesis. In humans, GLDC is part of a multienzyme complex (which includes the lipoyl-containing H-protein) that couples the decarboxylation of glycine to the biosynthesis of serine. Details of the GLDC-catalyzed glycine decarboxylation reaction are critical to drug development but remain elusive. This is the first report on the mechanism of the GLDC-catalyzed reaction and shows that GLDC is an unusual PLP-containing α-amino acid decarboxylase that removes carbon dioxide from the glycine substrate without releasing the expected amine (methylamine, a metabolic precursor of toxic formaldehyde) as a product. In an unusual decarboxylation mechanism, the resulting aminomethyl moiety is instead transferred to an accessory H-protein. This study defines the role of H-protein in GLDC-catalyzed glycine decarboxylation. (1) H-Protein is not required for glycine decarboxylation but, instead, is required for the release of the aminomethyl moiety from the quinonoid adduct. (2) Glycine decarboxylation is reversible and presumably proceeds through a stable quinonoid intermediate. (3) The physiological product of glycine decarboxylation is H-protein-S-aminomethyl dihydrolipoyllysine and not methylamine (in the absence of H-protein, the aminomethyl moiety remains as a quinonoid adduct). Mechanistic insights obtained from this study will inform future efforts for targeted anticancer therapeutic development.

High-resolution X-ray crystal structure of bovine H-protein at 0.88 A resolution.

Recent technical improvements in macromolecular X-ray crystallography have significantly improved the resolution limit of protein structures. However, examples of high-resolution structure determination are still limited. In this study, the X-ray crystal structure of bovine H-protein, a component of the glycine cleavage system, was determined at 0.88 A resolution. This is the first ultrahigh-resolution structure of an H-protein. The data were collected using synchrotron radiation. Because of limitations of the hardware, especially the dynamic range of the CCD detector, three data sets (high-, medium- and low-resolution data sets) were measured in order to obtain a complete set of data. To improve the quality of the merged data, the reference data set was optimized for merging and the merged data were assessed by comparing merging statistics and R factors against the final model and the number of visualized H atoms. In addition, the advantages of merging three data sets were evaluated. The omission of low-resolution reflections had an adverse effect on visualization of H atoms in hydrogen-omit maps. Visualization of hydrogen electron density is a good indicator for assessing the quality of high-resolution X-ray diffraction data.

Alternative splicing produces an H-protein with better substrate properties for the P-protein of glycine decarboxylase.

Several thousand plant genes are known to produce multiple transcripts, but the precise function of most of the alternatively encoded proteins is not known. Alternative splicing has been reported for the H-protein subunit of glycine decarboxylase in the genus Flaveria. H-protein has no catalytic activity itself but is a substrate of the three enzymatically active subunits, P-, T- and L-protein. In C(4) species of Flaveria, two H-proteins originate from single genes in an organ-dependent manner. Here, we report on differences between the two alternative H-protein variants with respect to their interaction with the glycine-decarboxylating subunit, P-protein. Steady-state kinetic analyses of the alternative Flaveria H-proteins and artificially produced 'alternative' Arabidopsis H-proteins, using either pea mitochondrial matrix extracts or recombinant cyanobacterial P-protein, consistently demonstrate that the alternative insertion of two alanine residues at the N-terminus of the H-protein elevates the activity of P-protein by 20%in vitro, and could promote glycine decarboxylase activity in vivo.

Protein disorder is positively correlated with gene expression in Escherichia coli.

We considered, on a global scale, the relationship between the predicted fraction of protein disorder and the RNA and protein expression in Escherichia coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins.

Mitochondria from leaf mesophyll cells of C(4) plants are deficient in the H protein of glycine decarboxylase complex.

Mesophyll mitochondria from green leaves of the C(4) plants Zea mays (NADP-ME-type), Panicum miliaceum (NAD-ME-type) and Panicum maximum (PEP-CK-type) oxidized NADH, malate and succinate at relatively high rates with respiratory control, but glycine was not oxidized. Among the mitochondrial proteins involved in glycine oxidation, the L, P and T proteins of glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) were present, while the H protein of GDC was undetectable. In contrast, mesophyll mitochondria from etiolated leaves of Z. mays oxidized glycine at a slow rate and with no respiratory control, and contained the H protein as well as the other GDC proteins and SHMT. The T and P proteins and SHMT were present in the mitochondria from etiolated leaves at significantly higher levels than in those from green leaves of Z. mays. The content of the L protein was almost identical in all three C(4) plants examined and close to the value obtained for mesophyll mitochondria from the C(3) plant Pisum sativum, whereas the other GDC proteins and SHMT were less abundant than the L protein. We discuss possible reasons for the H protein's absence in mesophyll mitochondria of C(4) plants, as well as the role(s) the other GDC components could play in its absence.

Properties of recombinant glycine decarboxylase P- and H-protein subunits from the cyanobacterium Synechocystis sp. strain PCC 6803.

The multi-enzyme complex glycine decarboxylase is important for one-carbon metabolism, essential for the photorespiratory glycolate cycle of plants, and comprises four different polypeptides, P-, H-, T-, and L-protein. We report on the production and properties of recombinant P-protein from the cyanobacterium Synechocystis and also describe features of recombinant H-protein from the same organism. The P-protein shows enzymatic activity with lipoylated H-protein and very low activity with H-apoprotein or lipoate as artificial cofactors. Its affinity towards glycine is unaffected by the presence and nature of the methyleneamine acceptor molecule. The cyanobacterial H-protein apparently forms stable dimers.