Glutathione biosynthesis

A reusable two-reaction pathway that converts L-glutamate, L-cysteine, and glycine to glutathione through gamma-L-glutamyl-L-cysteine. The module represents the conserved GshA/GCLC and GshB/GSS reaction roles independently of genomic arrangement or enzyme regulation. Glutathione reduction, conjugation, peroxide detoxification, degradation, and the wider gamma-glutamyl cycle are outside the boundary.

MODULE:glutathione_biosynthesisCOMPLETEABSTRACTMetabolic Pathwaymodules/glutathione_biosynthesis.yaml

glutathione biosynthetic processGO:0006750

GO:0006750

glutathione biosynthetic process

GO:0006750 provides the biological-process context.

RHEA:13285

glutamate--cysteine ligase reaction

RHEA:13285 defines ATP-dependent formation of gamma-L-glutamyl-L-cysteine from L-glutamate and L-cysteine.

RHEA:13557

glutathione synthetase reaction

RHEA:13557 defines ATP-dependent addition of glycine to gamma-L-glutamyl-L-cysteine to form glutathione.

UniProtKB:Q9CM00

Pasteurella multocida bifunctional glutathione biosynthesis protein GshAB

The reviewed GshAB/GshF record assigns both glutamate--cysteine ligase and glutathione synthetase reactions (by HAMAP rule MF_00782), a fused implementation of the same two ordered reaction roles whose experimental basis is PMID:16339152.

PMID:16339152

Characterization of the bifunctional gamma-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida

Recombinant characterization establishes the two-domain GshF fusion as a bifunctional implementation of both ordered glutathione-biosynthesis reactions.

file:human/GCLC/GCLC-ai-review.yaml

Human GCLC gene review

P48506 provides a eukaryotic exemplar for the first catalytic reaction.

file:ECOLI/gshA/gshA-ai-review.yaml

E. coli gshA gene review

P0A6W9 is the purified, kinetically characterized bacterial GshA and the PAINT seed of PTN002215154 (PTHR38761).

file:ECOLI/gshB/gshB-ai-review.yaml

E. coli gshB gene review

P04425 is the purified homotetrameric bacterial GshB and the PAINT seed of PTN000492686 (PTHR21621:SF4); the gshB mutant accumulates gamma-glutamylcysteine.

file:PASMU/gshAB/gshAB-ai-review.yaml

P. multocida gshAB gene review

Q9CM00 performs both reactions on one polypeptide; its N-terminal domain is PTHR38761:SF1 and its C-terminal ATP-grasp domain is IPR040657.

PMID:9895302

Overexpression of the regulatory subunit of gamma-glutamylcysteine synthetase in HeLa cells increases gamma-glutamylcysteine synthetase activity and confers drug resistance.

Experimental addition and overexpression of the human regulatory subunit increased GCL activity, supporting its treatment as an accessory modifier rather than a separate catalytic reaction in the pathway.

file:human/GSS/GSS-ai-review.yaml

Human GSS gene review

P48637 provides a eukaryotic exemplar for the second catalytic reaction.

file:modules/glutathione_biosynthesis-deep-research-openscientist.md

OpenScientist research for glutathione biosynthesis

The generic review evaluates the two conserved reaction roles, pathway boundary, family assignments, and lineage-dependent enzyme forms.

file:PSEPK/gshA/gshA-ai-review.yaml

PSEPK gshA gene review

Q88R90 supplies the glutamate--cysteine ligase step.

file:PSEPK/gshB/gshB-ai-review.yaml

PSEPK gshB gene review

Q88D35 supplies the glutathione synthetase step.

PMID:40302248

High-Throughput Tn-Seq Screens Identify Both Known and Novel Pseudomonas putida KT2440 Genes Involved in Metal Tolerance.

A P. putida KT2440 Tn-seq study identifies PP_0243 as gshA, describes its glutamate--cysteine ligase reaction, and reports reduced mutant fitness under copper and cobalt stress; gshB PP_4993 showed a weaker copper phenotype that did not pass the study's statistical threshold.

file:PSEPK/PP_3253/PP_3253-ai-review.yaml

PSEPK PP_3253 gene review

Q88HV0 is a distinct type-2/YbdK-family ligase candidate with no established physiological contribution to KT2440 glutathione synthesis.

PMID:15211520

YbdK is a carboxylate-amine ligase with a gamma-glutamyl:Cysteine ligase activity: crystal structure and enzymatic assays.

Purified E. coli YbdK has weak glutamate--cysteine ligase activity in vitro, supporting candidate status for this family but not replacement of canonical GshA in the reusable pathway.

YeastPathways:GLUTATHIONESYN-PWY

glutathione biosynthesis (S. cerevisiae, SGD YeastPathways)

YeastCyc lists GLUTCYSLIG-RXN (GSH1) and GLUTATHIONE-SYN-RXN (GSH2); both assignments were retained.

PMID:12702279

An overview on glutathione in Saccharomyces versus non-conventional yeasts.

Review of glutathione synthesis and functions in S. cerevisiae and other yeasts.

PMID:8824231

Identification of the structural gene for glyoxalase I from Saccharomyces cerevisiae.

gsh1 mutants make no glutathione, whereas gsh2 mutants accumulate the gamma-glutamylcysteine intermediate, consistent with the two-step order.

The gsh1-deficient mutant, which could not produce glutathione at all ... The gsh2-deficient mutant, which accumulates gamma-glutamylcysteine (an intermediate of glutathione biosynthesis)

3Nodes

2Parts

0Variant Sets

0Variants

3Annotons

1Connections

Derived QC

Recommended-field compliance

100.0% recommended fields populated

All recommended fields populated.

Module deep research

✓ present

glutathione_biosynthesis-deep-research-openscientist.md (openscientist)

Leaf nodes lacking representative members

✓ representative grounding skipped for abstract module.

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

12 gene-function check(s) reflected in core functions · 0 conflict(s) · 1 evidence or coverage gap(s) (17 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

Annoton	Participant	Function	Assessment
gshA_activity	gshA (Pseudomonas putida KT2440) UniProtKB:Q88R90 Family Representative	glutamate-cysteine ligase activity GO:0004357	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshA_activity	gshA (Escherichia coli K-12) UniProtKB:P0A6W9 Family Representative	glutamate-cysteine ligase activity GO:0004357	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshA_activity	GCLC (human) UniProtKB:P48506 Family Representative	glutamate-cysteine ligase activity GO:0004357	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshA_activity	gshAB / gshF (Pasteurella multocida) UniProtKB:Q9CM00 Family Representative	glutamate-cysteine ligase activity GO:0004357	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshA_activity	GSH1 (Saccharomyces cerevisiae) UniProtKB:P32477 Family Representative	glutamate-cysteine ligase activity GO:0004357	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshA_activity	gcs1 (Schizosaccharomyces pombe) UniProtKB:Q09768 Family Representative	glutamate-cysteine ligase activity GO:0004357	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshB_activity	gshB (Pseudomonas putida KT2440) UniProtKB:Q88D35 Family Representative	glutathione synthase activity GO:0004363	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshB_activity	gshB (Escherichia coli K-12) UniProtKB:P04425 Family Representative	glutathione synthase activity GO:0004363	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshB_activity	GSS (human) UniProtKB:P48637 Family Representative	glutathione synthase activity GO:0004363	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshB_activity	GSH2 (Saccharomyces cerevisiae) UniProtKB:Q08220 Family Representative	glutathione synthase activity GO:0004363	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshB_activity	gsa1 (Schizosaccharomyces pombe) UniProtKB:P35669 Family Representative	glutathione synthase activity GO:0004363	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshAB_glutathione_synthetase_activity	gshAB / gshF (Pasteurella multocida) UniProtKB:Q9CM00 Family Representative	glutathione synthase activity GO:0004363	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
gshA_activity	glutamate--cysteine ligases PANTHER:PTHR38761:SF1 Family	glutamate-cysteine ligase activity GO:0004357	Family Supported Family review supports GO:0004357 FAMILY_WIDE. Held by monofunctional GshA and by the N-terminal domain of GshAB alike; selected by the glutathione_biosynthesis module.
gshA_activity	glutamate--cysteine ligases PANTHER:PTHR11164:SF0 Family	glutamate-cysteine ligase activity GO:0004357	Family Supported Family review supports GO:0004357 FAMILY_WIDE. Asserted by glutathione_synthesis_gamma_glutamyl_cycle; accepted by GSH1, gcs1 and GCLC.
gshB_activity	glutathione-forming ATP-grasp domains PANTHER:PTHR21621:SF4 Family	glutathione synthase activity GO:0004363	Family Supported Family review supports GO:0004363 for PANTHER:PTHR21621:SF4. Bacterial GshB (SF4) only. All 53 SF4 members in the inventory are named glutathione synthetase, so no member exceptions are needed; other subfamilies ligate glutamate to different acceptors. Selected by the glutathione_biosynthesis module.
gshB_activity	glutathione-forming ATP-grasp domains PANTHER:PTHR11130:SF0 Family	glutathione synthase activity GO:0004363	Family Supported Family review supports GO:0004363 FAMILY_WIDE. Asserted by glutathione_synthesis_gamma_glutamyl_cycle; accepted by GSH2, gsa1, GSS and rat Gss.
gshAB_glutathione_synthetase_activity	GshAB/GshF C-terminal ATP-grasp glutathione synthetase domains InterPro:IPR040657 Family	glutathione synthase activity GO:0004363	Family Review Missing No supported structured family review is available for InterPro:IPR040657.

Gene-review completeness (11/11 grounded genes reviewed)

11 complete review(s) · 5 with deep research · 0 missing review · 6 reviewed but lacking deep research

Gene	Review	Complete	Deep research
GCLC P48506	✓	✓	✓
gcs1 Q09768	✓	✓	✗
gsa1 P35669	✓	✓	✗
GSH1 P32477	✓	✓	✓
GSH2 Q08220	✓	✓	✓
gshA P0A6W9	✓	✓	✗
gshA Q88R90	✓	✓	✗
gshAB Q9CM00	✓	✓	✗
gshB P04425	✓	✓	✗
gshB Q88D35	✓	✓	✓
GSS P48637	✓	✓	✓

Tree

Glutathione biosynthesisMetabolic Pathwayglutathione_biosynthesis

Parts
1. 1. gamma-L-glutamyl-L-cysteine formation
2. Glutamate--cysteine ligaseReactionglutamate_cysteine_ligase_step
  
  Annotons
  - Glutamate--cysteine ligase Family: glutamate--cysteine ligases
3. 2. glutathione formation
4. Glutathione synthetaseReactionglutathione_synthetase_step
  
  Annotons
  - Glutathione synthetase Family: glutathione-forming ATP-grasp domains
  - GshAB/GshF glutathione synthetase domain Family: GshAB/GshF C-terminal ATP-grasp glutathione synthetase domains

Details

Glutathione biosynthesisMetabolic Pathwayglutathione_biosynthesis

glutathione biosynthetic processGO:0006750

Exact bacterial and human UniProt exemplars delimit both reaction roles across prokaryotic GshA/GshB and eukaryotic GCLC/GSS families. The narrow subfamilies are used instead of parent families that include ligases with other substrates. The same reviewed GshAB/GshF exemplar appears under both leaf activities because a single bifunctional polypeptide can implement both ordered reactions; it does not collapse the pathway into one biological step. PANTHER classifies the whole GshAB/GshF fusion as PTHR38761:SF1 through its first activity, but that family is deliberately not asserted as a selector for the second leaf because 104 of its 119 members are monofunctional GshA (the PTHR38761 family review leaves GO:0004363 UNRESOLVED for that reason). The second leaf therefore has two annotons: the GshB/GSS families, and a separate GshAB/GshF annoton selected by IPR040657 and supported by PMID:16339152. Mammalian GCLM can modify GCLC kinetics but is not a third catalytic reaction and is therefore not a required module part. Type 2/YbdK-family ligases are candidate alternatives outside the required chain unless organism-specific physiological evidence establishes that they implement the first reaction. PSEPK PP_3253/Q88HV0 maps to PANTHER PTHR36510:SF1. Reviewed E. coli YbdK/P77213, independently verified in the same subfamily, is the source of the weak in-vitro family-level activity; it does not establish a glutathione-biosynthesis role for Q88HV0. No molecular function or location is duplicated at module level. Four PAINT IBD nodes are asserted, each verified in its family review: PTN002215154 (PTHR38761, seeded by E. coli GshA) and PTN000126115 (PTHR11164, as in maintainer PR #5076) for the first step, and PTN000492686 (PTHR21621, seeded by E. coli GshB) and PTN000122964 (PTHR11130) for the second. The module is species-neutral (ABSTRACT): the same two reactions are implemented by unrelated bacterial and eukaryotic enzyme families, with E. coli, P. putida KT2440, P. multocida, human and yeast exemplars that carry experimental or curated evidence. ALIGN (YeastPathways project, 2026-10): S. cerevisiae GSH1/GSH2 and S. pombe gcs1/gsa1 were added as eukaryotic exemplars; each falls in the same PANTHER subfamily as human GCLC (PTHR11164:SF0) or GSS (PTHR11130:SF0). S. pombe also has a GCLM-like modifier (gcs2, O94246, PTHR13295) that the PomBase model links as a positive regulator of gcs1; S. cerevisiae has no GCLM ortholog, so the modifier remains outside the required chain. Both yeast GO-CAMs agree with the two-step order and cytosolic location.

Connections

glutamate_cysteine_ligase_step -> glutathione_synthetase_step Provides Input For

GshA/GCLC supplies gamma-L-glutamyl-L-cysteine to GshB/GSS.

Part 1: gamma-L-glutamyl-L-cysteine formation

Glutamate--cysteine ligaseReactionglutamate_cysteine_ligase_step

Annotons

Glutamate--cysteine ligase

gshA_activity

Participant: Family: glutamate--cysteine ligases

Family:

glutamate--cysteine ligases

Representative Members: gshA (Pseudomonas putida KT2440)UniProtKB:Q88R90 gshA (Escherichia coli K-12)UniProtKB:P0A6W9 GCLC (human)UniProtKB:P48506 gshAB / gshF (Pasteurella multocida)UniProtKB:Q9CM00 GSH1 (Saccharomyces cerevisiae)UniProtKB:P32477 gcs1 (Schizosaccharomyces pombe)UniProtKB:Q09768

Function

glutamate-cysteine ligase activityGO:0004357

Substrates: L-glutamate L-cysteine ATP

Products: gamma-L-glutamyl-L-cysteine ADP phosphate proton

Forms the gamma-glutamyl peptide bond in the first committed glutathione-biosynthesis reaction.

Part 2: glutathione formation

Glutathione synthetaseReactionglutathione_synthetase_step

Annotons

Glutathione synthetase

gshB_activity

Participant: Family: glutathione-forming ATP-grasp domains

Family:

glutathione-forming ATP-grasp domains

Representative Members: gshB (Pseudomonas putida KT2440)UniProtKB:Q88D35 gshB (Escherichia coli K-12)UniProtKB:P04425 GSS (human)UniProtKB:P48637 GSH2 (Saccharomyces cerevisiae)UniProtKB:Q08220 gsa1 (Schizosaccharomyces pombe)UniProtKB:P35669

Function

glutathione synthase activityGO:0004363

Substrates: gamma-L-glutamyl-L-cysteine glycine ATP

Products: glutathione ADP phosphate proton

Adds glycine to gamma-L-glutamyl-L-cysteine to complete glutathione synthesis.

GshAB/GshF glutathione synthetase domain

gshAB_glutathione_synthetase_activity

Participant: Family: GshAB/GshF C-terminal ATP-grasp glutathione synthetase domains

Family:

GshAB/GshF C-terminal ATP-grasp glutathione synthetase domains

Representative Members: gshAB / gshF (Pasteurella multocida)UniProtKB:Q9CM00

Function

glutathione synthase activityGO:0004363

Substrates: gamma-L-glutamyl-L-cysteine glycine ATP

Products: glutathione ADP phosphate proton

Alternative implementation of the glycine-adding step on the same polypeptide as the GshA-like domain, in GshF-type bacteria such as Pasteurella, Streptococcus and Listeria.