scholarly journals Improvement of glycine biosynthesis from one‐carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro

2021 ◽  
Author(s):  
Yingying Xu ◽  
Jie Ren ◽  
Wei Wang ◽  
An‐Ping Zeng
Keyword(s):  
Glycine Cleavage System ◽  
Carbon Compounds ◽  
Glycine Cleavage

scholarly journals Stand-alone lipoylated H-protein of the glycine cleavage system enables glycine cleavage and the synthesis of glycine from one-carbon compounds in vitro

2021 ◽  
Author(s):  
Yingying Xu ◽  
Yuchen Li ◽  
Han Zhang ◽  
Jinglei Nie ◽  
Jie Ren ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
The Other ◽  
Glycine Cleavage System ◽  
Carbon Compounds ◽  
Individual Reaction ◽  
L Proteins ◽  
Glycine Cleavage ◽  
The Individual ◽  
H Protein

H-protein, one of the four component proteins (H, T, P and L) of glycine cleavage system (GCS), is generally considered a shuttle protein interacting with the other three GCS-proteins via a lipoyl swinging arm. We report that without P-, T- and L-proteins, lipoylated H-protein (Hlip) enables GCS reactions in both glycine cleavage and synthesis directions in vitro. This apparent catalytic activity is closely related to the cavity on the H-protein surface where the lipoyl arm is attached. Heating or mutation of selected residues in the cavity destroys or reduces the stand-alone activity of Hlip, which can be restored by adding the other three GCS-proteins. Systematic study of the Hlip-catalyzed overall GCS reactions and the individual reaction steps provides a first step towards understanding the stand-alone function of Hlip. The results in this work provide some inspiration for further understanding the mechanism of the GCS and give some interesting implications on the evolution of the GCS.

Cysteamine inhibition of [15N]-glycine turnover in cystinosis and of the glycine cleavage system in vitro

Metabolism ◽  
1981 ◽  
Vol 30 (11) ◽  
pp. 1096-1103 ◽  
Author(s):  
Marc Yudkoff ◽  
Itzhak Nissim ◽  
Adele Schneider ◽  
Stanton Segal
Keyword(s):  
Glycine Cleavage System ◽  
Glycine Cleavage

scholarly journals Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-prodanediol

2020 ◽  
Author(s):  
Yingying Xu ◽  
Hao Meng ◽  
Jie Ren ◽  
An-Ping Zeng
Keyword(s):  
Release Rate ◽  
Metabolic Pathway ◽  
Protein Concentration ◽  
Biosynthetic Pathway ◽  
Oxidative Degradation ◽  
Glycine Cleavage System ◽  
Speed Up ◽  
Glycine Cleavage ◽  
H Protein

Abstract Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

scholarly journals Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-prodanediol

2020 ◽  
Author(s):  
Yingying Xu ◽  
Hao Meng ◽  
Jie Ren ◽  
An-Ping Zeng
Keyword(s):  
Release Rate ◽  
Metabolic Pathway ◽  
Protein Concentration ◽  
Biosynthetic Pathway ◽  
Oxidative Degradation ◽  
Glycine Cleavage System ◽  
Speed Up ◽  
Glycine Cleavage ◽  
H Protein

Abstract Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

scholarly journals Tracing Metabolic Fate of Mitochondrial Glycine Cleavage System Derived Formate In Vitro and In Vivo

2020 ◽  
Vol 21 (22) ◽  
pp. 8808
Author(s):  
Yee-Ling Tan ◽  
Nga-Lai Sou ◽  
Feng-Yao Tang ◽  
Hsin-An Ko ◽  
Wei-Ting Yeh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hepatoma Cell ◽  
Model Systems ◽  
Whole Body ◽  
Co2 Production ◽  
Glycine Cleavage System ◽  
Metabolic Fate ◽  
Glycine Cleavage

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.

scholarly journals Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-prodanediol

2020 ◽  
Author(s):  
Yingying Xu ◽  
Hao Meng ◽  
Jie Ren ◽  
An-Ping Zeng
Keyword(s):  
Release Rate ◽  
Metabolic Pathway ◽  
Protein Concentration ◽  
Biosynthetic Pathway ◽  
Oxidative Degradation ◽  
Glycine Cleavage System ◽  
Speed Up ◽  
Glycine Cleavage ◽  
H Protein

Abstract Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO 2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N 5 ,N 10 -methylene-tetrahydrofolate (5,10-CH 2 -THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH 2 -THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (H int ) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (H red ), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by H int . Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro . This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

scholarly journals Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol

2020 ◽  
Vol 14 (1) ◽  
Author(s):  
Yingying Xu ◽  
Hao Meng ◽  
Jie Ren ◽  
An-Ping Zeng
Keyword(s):  
Release Rate ◽  
Metabolic Pathway ◽  
Protein Concentration ◽  
Biosynthetic Pathway ◽  
Oxidative Degradation ◽  
Glycine Cleavage System ◽  
Speed Up ◽  
Glycine Cleavage ◽  
H Protein

AbstractGlycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

scholarly journals Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria

1994 ◽  
Vol 302 (1) ◽  
pp. 223-228 ◽  
Author(s):  
F Rebeille ◽  
M Neuburger ◽  
R Douce
Keyword(s):  
Dissociation Constants ◽  
Oxidative Degradation ◽  
Maximal Activity ◽  
Serine Hydroxymethyltransferase ◽  
In Vitro Experiments ◽  
Glycine Cleavage System ◽  
Kd Values ◽  
Bulk Medium ◽  
Glycine Cleavage

The aim of the present work was to further determine how the T-protein of the glycine-cleavage system and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the glycine-cleavage system was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.

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