Definition of L-proline biosynthesis
As rules and steps, or see full text
Rules
Overview: Proline biosynthesis in GapMind is based on MetaCyc pathways L-proline biosynthesis I from L-glutamate (link) and III via L-ornithine aminotransferase (link). GapMind also describes proline synthesis via ornithine cyclodeaminase (PMID:11489875). Pathway II from arginine (link) should perhaps be included, but is not; it is reported in nitrogen-fixing cyanobacteria and the bacteria it was reported in also have pathway I. Pathway IV is not included because it is not reported in prokaryotes (link). Also, pathway III is described in MetaCyc as occuring only in plants, but it has been reported in the archaeon Thermococcus kodakarensis (PMC5846162).
- all:
- proB, proA and proC
- or ornithine, OAT and proC
- or ornithine and cyclodeaminase
- Comment: Pathway I is proBAC. Pathway II involves ornithine aminotransferase (OAT). Some methanogens synthesize proline via ornithine cyclodeaminase (PMID:11489875).
- ornithine:
- argA, argB, argC, argD and argE
- or argJ, argB, argC and argD
- or lysW, argX, lysZ, lysY, lysJ and lysK
- Comment: In L-arginine biosynthesis I, ornithine forms via acetylated intermediates, argA, and argE (link). In L-arginine biosynthesis II, ornithine forms via acetylated intermediates and argJ (link). In L-arginine biosynthesis IV, ornithine forms via LysW-modified intermediates (link).
Steps
lysW: 2-aminoadipate/glutamate carrier protein
- Curated proteins matching alpha-aminoadipate%carrier
- UniProt sequence Q5JFV9: SubName: Full=Probable lysine biosynthesis protein {ECO:0000313|EMBL:BAD84468.1};
- Comment: LysW is a carrier protein for intermediates in lysine and/or ornithine biosynthesis. It is sometimes called the amino group carrier protein. TK0279 (Q5JFV9) from Thermococcus kodakarensis was characterized, see PMC5076833.
- Total: 5 characterized proteins
argA: N-acylglutamate synthase
- Curated proteins or TIGRFams with EC 2.3.1.1
- Curated proteins matching N-succinylglutamate synthase
- UniProt sequence Q8A1A5_BACTN: RecName: Full=GNAT family N-acetyltransferase {ECO:0008006|Google:ProtNLM};
- UniProt sequence L0G3H4_ECHVK: SubName: Full=Acetyltransferase, N-acetylglutamate synthase {ECO:0000313|EMBL:AGA80057.1};
- Curated sequence A0A0H2X8L7: acetylglutamate kinase (EC 2.7.2.8)
- Curated sequence Q8P8J6: acetylglutamate kinase (EC 2.7.2.8)
- Ignore hits to G6759-MONOMER when looking for 'other' hits (L-amino acid N-acyltransferase MnaT; L-methionine N-acyltransferase; L-methionine sulfoximine/L-methionine sulfone N-acetyltransferase; L-phenylglycine N-acetyltransferase; EC 2.3.1.-. L-amino acid N-acyltransferase (EC 2.3.1.1). L-amino acid N-acyltransferase (EC 2.3.1.1))
- Ignore hits to items matching EC 2.3.1.35 when looking for 'other' hits
- Ignore hits to CH_123299 when looking for 'other' hits (arginine biosynthetic enzyme activities)
- Curated sequence CH_122594: glutamate N-acetyltransferase (Eurofung)
- Ignore hits to Q9I3W7 when looking for 'other' hits (amino-acid N-acetyltransferase (EC 2.3.1.1))
- Ignore hits to Q8P8J6 when looking for 'other' hits (acetylglutamate kinase (EC 2.7.2.8))
- Predicted: UniProt sequence A0A127FCT3: RecName: Full=DUF1611 domain-containing protein {ECO:0008006|Google:ProtNLM};
- Comment: Bacteroidetes have a divergent N-acylglutamate synthase, see BT3761 (Q8A1A5_BACTN) or Echvi_3845 (L0G3H4_ECHVK). Bacteroides use succinylated intermediates (PMID:16704984), so their proteins are probably N-succinylglutamate synthases. (These enzymes are also known as argA2, see PMC9026213.) Q8P8J6 is annotated as argB in BRENDA, but it is also argA (a fusion protein). N515DRAFT_3768 (A0A1I2DIM7) is similar to ArgAB fusion proteins and mutants are rescued by arginine. It is not clear if mnaT (link) would acetylate arginine, so it is ignored. Some EC 2.3.1.35 enzymes are probably dedicated for converting N-acetylornithine back to ornithine, while others are bifunctional for N-acetylglutamate formation as well (argJ); so similarity to this EC number is inconclusive. For some CharProtDB entries it is unclear if they have this activity or not, while others are labeled with this function but without the EC number. Q9I3W7 is annotated as this in BRENDA but we did not find experimental evidence of its activity on glutamate, so it is ignored. In Steroidobacter denitrificans, argA is missing but the arginine synthesis cluster includes a potential N-acetyltransferase ACG33_RS14135 (A0A127FCT3). Homologs of this protein are conserved in the cluster, and it is never found together with argA; it is distantly related to D-glutamate acetyltransferase dgcN (formerly DUF1611, see PMC10030869).
- Total: 1 HMMs and 32 characterized proteins
argB: N-acylglutamate kinase
- Curated proteins or TIGRFams with EC 2.7.2.8
- Curated proteins matching N-succinylglutamate kinase
- Ignore hits to O67848 when looking for 'other' hits (UDP-3-O-acyl-N-acetylglucosamine deacetylase (EC 3.5.1.108))
- Ignore hits to Q87EL2 when looking for 'other' hits (amino-acid N-acetyltransferase (EC 2.3.1.1))
- Curated sequence CH_123299: arginine biosynthetic enzyme activities
- Comment: ArgB includes Bacteroides proteins that act on N-succinylglutamate instead of the usual N-acetylglutamate (i.e. BT3395). See "Discovery of novel pathways of microbial arginine biosynthesis" (2010), PhD thesis of Juan Manuel Cabrera Luque, which shows that argB from B. fragilis is N-succinylglutamate kinase. O67848 in BRENDA seems likely to be argB but is misannotated as a deacetylase; also, Q87EL2 is likely to be argB as well as argA. CH_123299 has a broader annotation but probably has this activity.
- Total: 1 HMMs and 18 characterized proteins
argC: N-acylglutamylphosphate reductase
- Curated proteins or TIGRFams with EC 1.2.1.38
- Curated proteins matching N-succinylglutamylphosphate reductase
- Curated sequence CH_123299: arginine biosynthetic enzyme activities
- UniProt sequence E4PLW0: RecName: Full=N-acetyl-gamma-glutamyl-phosphate reductase {ECO:0000256|HAMAP-Rule:MF_00150}; Short=AGPR {ECO:0000256|HAMAP-Rule:MF_00150}; EC=1.2.1.38 {ECO:0000256|HAMAP-Rule:MF_00150}; AltName: Full=N-acetyl-glutamate semialdehyde dehydrogenase {ECO:0000256|HAMAP-Rule:MF_00150}; Short=NAGSA dehydrogenase {ECO:0000256|HAMAP-Rule:MF_00150};
- Comment: ArgC includes Bacteroides proteins that probably act on N-succinylglutamylphosphate instead of N-acetylglutamylphosphate (i.e. BT3759). CH_123299 has a broader annotation but probably has this activity. HP15_352 (E4PLW0) from Marinobacter adhaerens HP15 is important for fitness in most minimal media, unless arginine is provided.
- Total: 2 HMMs and 15 characterized proteins
argD: N-acetylornithine aminotransferase
- Curated proteins or TIGRFams with EC 2.6.1.11
- Ignore hits to items matching EC 2.6.1.81 when looking for 'other' hits
- Ignore hits to items matching EC 2.6.1.19 when looking for 'other' hits
- Ignore hits to MONOMER-18314 when looking for 'other' hits ([amino group carrier protein]-C-terminal-L-glutamyl-γ-L-lysine aminotransferase (EC 2.6.1.118; EC 2.6.1.124))
- Ignore hits to items matching EC 2.6.1.48 when looking for 'other' hits
- UniProt sequence A0A806JQF3: RecName: Full=Acetylornithine aminotransferase {ECO:0000256|HAMAP-Rule:MF_01107}; Short=ACOAT {ECO:0000256|HAMAP-Rule:MF_01107}; EC=2.6.1.11 {ECO:0000256|HAMAP-Rule:MF_01107};
- Comment: This aminotransferase for converting N-acetylglutamate semialdehyde to acetylornithine is often similar to succinylornithine transaminases (EC 2.6.1.81), 4-aminobutyrate aminotransferases (EC 2.6.1.19), or 5-aminovalerate transaminases (EC 2.6.1.48). (Succinylornithine and 4-aminobutyrate transaminases are also reported to be active on N-acetylornithine and this seems likely for 5-aminovalerate transaminases as well.) link is given this EC nmber but is actually LysW-lysine/ornithine aminotransferase (LysJ). PMID:A0A806JQF3 show that Rv1655 (A0A806JQF3) from Mycobacterium tuberculosis is argD.
- Total: 14 characterized proteins
argE: N-acetylornithine deacetylase
- Curated proteins or TIGRFams with EC 3.5.1.16
- Curated proteins matching N-acetylcitrulline deacetylase
- Predicted: UniProt sequence W3Y6L2: SubName: Full=Amidohydrolase {ECO:0000313|EMBL:ETS93161.1};
- Comment: This EC number also includes N-acetylcitrulline deacetylase, which is part of pathway III. A putative amidohydrolase "ArgA3" (W3Y6L2) is in a conserved operon with ornithine carbamoyltransferase (the next step) in Veillonella and related genera (PMC9026213). The genome context includes other arginine synthesis genes as well. Although ArgA3 was proposed to be a N-acetylglutamate synthase, it is related to N-acetyl-cysteine deacetylase, and the genomes with "ArgA3" are lacking argE as well; so we think argE is the more likely function.
- Total: 1 HMMs and 7 characterized proteins
argJ: ornithine acetyltransferase
- Curated proteins or TIGRFams with EC 2.3.1.35
- Ignore hits to CH_122594 when looking for 'other' hits (glutamate N-acetyltransferase (Eurofung))
- Comment: In pathway II (acetyl cycle), instead of an acetylornithine deacetylase, the acetyltransferase argJ converts N-acetylornithine to ornithine. ArgJ may also form N-acetylglutamate (replacing argA). CH_122594 lacks an EC number (not fully characterized) and is likely to be ArgJ (50% identity to O94346)
- Total: 16 characterized proteins
argX: glutamate--LysW ligase
- Curated proteins or TIGRFams with EC 6.3.2.60
- Curated sequence Q970U6: Glutamate--LysW ligase ArgX; EC 6.3.2.-
- Ignore hits to items matching 6.3.2.43 when looking for 'other' hits
- Comment: There is also an archaeal pathway from glutamate to ornithine with LysW as the carrier protein, instead of N-acyl intermediates. This pathway is analogous to the conversion of alpha-aminoadipate to lysine, and many of the enzymes are bifunctional. But the initial ligation of LysW to arginine has a dedicated enzyme in many archaea. Q970U6 is given a generic EC number in SwissProt, but has this function. EC 6.4.2.43 (LysW-2-aminoadipate ligases) are ignored because some are bifunctional (PMC5076833).
- Total: 3 characterized proteins
lysZ: [LysW]-glutamate kinase
- Curated proteins or TIGRFams with EC 2.7.2.19
- UniProt sequence Q5JFW2: RecName: Full=Putative [LysW]-aminoadipate/[LysW]-glutamate kinase {ECO:0000256|HAMAP-Rule:MF_02082}; EC=2.7.2.17 {ECO:0000256|HAMAP-Rule:MF_02082}; EC=2.7.2.19 {ECO:0000256|HAMAP-Rule:MF_02082};
- Predicted: UniProt sequence D4GYN9: RecName: Full=Putative [LysW]-aminoadipate/[LysW]-glutamate kinase {ECO:0000256|HAMAP-Rule:MF_02082}; EC=2.7.2.17 {ECO:0000256|HAMAP-Rule:MF_02082}; EC=2.7.2.19 {ECO:0000256|HAMAP-Rule:MF_02082};
- Comment: TK0276 from Thermococcus kodakarensis (Q5JFW2) is bifunctional, for lysine and ornithine synthesis (PMC5076833). In Haloferax volcanii, the putative [LysW]-glutamate kinase (HVO_RS04915, D4GYN9) is in a conserved operon with LysW and with other arginine synthesis genes; this protein is more similar to a characterized [LysW]-2-aminoadipate 6-kinase, but haloarchaea do not use the aminoadipate pathway (PMC93780, PMC8305020).
- Total: 2 characterized proteins
lysY: [LysW]-glutamate-6-phosphate reductase
- Curated proteins or TIGRFams with EC 1.2.1.106
- UniProt sequence Q5JFW1: RecName: Full=Putative [LysW]-L-2-aminoadipate/[LysW]-L-glutamate phosphate reductase {ECO:0000255|HAMAP-Rule:MF_02083}; EC=1.2.1.103 {ECO:0000255|HAMAP-Rule:MF_02083}; EC=1.2.1.106 {ECO:0000255|HAMAP-Rule:MF_02083};
- Comment: TK0277 from Thermococcus kodakarensis (Q5JFW1) is bifunctional, for lysine and ornithine synthesis (PMC5076833).
- Total: 4 characterized proteins
lysJ: [LysW]-glutamate-semialdehyde aminotransferase
- Curated proteins or TIGRFams with EC 2.6.1.124
- Ignore hits to items matching 2.6.1.118 when looking for 'other' hits
- Comment: TK0275 from Thermococcus kodakarensis (Q5JFW3) is bifunctional, for lysine and ornithine synthesis (PMC5076833). Similarity to EC 2.6.1.118 ([LysW]-aminoadipate semialdehyde aminotransferase) is ignored because some are bifunctional (PMC5076833).
- Total: 1 characterized proteins
lysK: [LysW]-ornithine hydrolase
proB: glutamate 5-kinase
proA: gamma-glutamylphosphate reductase
proC: pyrroline-5-carboxylate reductase
OAT: L-ornithine 5-aminotransferase
- Curated proteins or TIGRFams with EC 2.6.1.13
- Curated sequence CH_124176: ornithine aminotransferase
- Ignore hits to O30508 when looking for 'other' hits (succinylornithine transaminase (EC 2.6.1.81). Succinylornithine transaminase/acetylornithine aminotransferase; ACOAT; SOAT; Succinylornithine aminotransferase; EC 2.6.1.11; EC 2.6.1.81. succinylornithine transaminase subunit (EC 2.6.1.13; EC 2.6.1.11; EC 2.6.1.81))
- Comment: Converts ornithine to glutamate semialdehyde. CH_124176 is annotated as ornithine transaminase but without the EC number; it probably has the same activity. BRENDA annotates AruC from Pseudomonas aeruginosa (O30508) has naving this activity as well, but it's probably not physiologically relevant and the Km is high (10 mM, PMID:238949); so O30508 considered an OAT in GapMind.
- Total: 1 HMMs and 26 characterized proteins
cyclodeaminase: ornithine cyclodeaminase
- Curated proteins or TIGRFams with EC 4.3.1.12
- UniProt sequence Q6LXX7: RecName: Full=LOR/SDH bifunctional enzyme conserved domain-containing protein {ECO:0000259|Pfam:PF04455};
- Comment: In many archaea, proline is formed from ornithine via a cyclodeaminase (PMID:11489875). Mls2 (Q6LXX7, MMP1218, MMP_RS06270) was identified as an ornithine cyclodeaminase (PMID:30636068), and it is important for fitness unless proline is added (Leslie Day).
- Total: 6 characterized proteins
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About GapMind
Each pathway is defined by a set of rules based on individual steps or genes. Candidates for each step are identified by using
ublast (a fast alternative to protein BLAST)
against a database of manually-curated proteins (most of which are experimentally characterized) or by using
HMMer with enzyme models (usually from
TIGRFam). Ublast hits may be split across two different proteins.
A candidate for a step is "high confidence" if either:
- ublast finds a hit to a characterized protein at above 40% identity and 80% coverage, and bits >= other bits+10.
- (Hits to curated proteins without experimental data as to their function are never considered high confidence.)
- HMMer finds a hit with 80% coverage of the model, and either other identity < 40 or other coverage < 0.75.
where "other" refers to the best ublast hit to a sequence that is not annotated as performing this step (and is not "ignored").
Otherwise, a candidate is "medium confidence" if either:
- ublast finds a hit at above 40% identity and 70% coverage (ignoring otherBits).
- ublast finds a hit at above 30% identity and 80% coverage, and bits >= other bits.
- HMMer finds a hit (regardless of coverage or other bits).
Other blast hits with at least 50% coverage are "low confidence."
Steps with no high- or medium-confidence candidates may be considered "gaps."
For the typical bacterium that can make all 20 amino acids, there are 1-2 gaps in amino acid biosynthesis pathways.
For diverse bacteria and archaea that can utilize a carbon source, there is a complete
high-confidence catabolic pathway (including a transporter) just 38% of the time, and
there is a complete medium-confidence pathway 63% of the time.
Gaps may be due to:
- our ignorance of proteins' functions,
- omissions in the gene models,
- frame-shift errors in the genome sequence, or
- the organism lacks the pathway.
GapMind relies on the predicted proteins in the genome and does not search the six-frame translation. In most cases, you can search the six-frame translation by clicking on links to Curated BLAST for each step definition (in the per-step page).
For more information, see:
If you notice any errors or omissions in the step descriptions, or any questionable results, please let us know
by Morgan Price, Arkin group, Lawrence Berkeley National Laboratory