Definition of L-isoleucine biosynthesis
As rules and steps, or see full text
Rules
Overview: Isoleucine biosynthesis in GapMind is based on MetaCyc pathways L-isoleucine biosynthesis I (from threonine) (link), II via citramalate (link), or IV from propanoate (link). These pathways share a common intermediate, 2-oxobutanoate, but vary in how the 2-oxobutanoate is formed. Pathway IV is included because propanoate is a common fermentative end product and need not be a nutrient requirement, but it is not always clear if it could be the main pathway to isoleucine. Pathway III (link), via glutamate mutase, is not included because the first step (glutamate mutase, EC 5.4.99.1) has not been linked to sequence and because no organism has been demonstrated to rely on this pathway to form oxobutanoate. Pathway V, from 2-methylbutanoate (link), is not included.
- all: oxobutanoate, ilvI, ilvH, ilvC, ilvD and ilvE
- oxobutanoate:
- ilvA
- or cimA, leuC, leuD and leuB
- or prpE, ofoa and ofob
- Comment: 2-oxobutanoate is formed by deaminating threonine (pathway I, ilvA), via citramalate synthase (pathway II, cimA), or via propionyl-CoA (pathway III, prpE)
Steps
ilvA: threonine deaminase
ilvH: acetohydroxybutanoate synthase catalytic subunit
ilvI: acetohydroxybutanoate synthase regulatory subunit
ilvC: 2-hydroxy-3-ketol-acid reductoisomerase
- Curated proteins or TIGRFams with EC 1.1.1.86
- Curated proteins or TIGRFams with EC 1.1.1.382
- Curated proteins or TIGRFams with EC 1.1.1.383
- Comment: The three EC numbers correspond to different preferences for NAD(P)H as the cofactor; the transformations to the carbon skeleton are the same.
- Total: 1 HMMs and 29 characterized proteins
ilvD: (R)-2,3-dihydroxy-3-methylpentanoate dehydratase
- Curated proteins or TIGRFams with EC 4.2.1.9
- Ignore hits to MONOMER-15882 when looking for 'other' hits (dihydroxy-acid dehydratase)
- Comment: The ignored enzyme is involved in salinosporamide A biosynthesis but does a very similar reaction and is >50% identical to N515DRAFT_0569, which is confirmed by fitness data to be biosynthetic
- Total: 1 HMMs and 6 characterized proteins
ilvE: isoleucine transaminase
cimA: (R)-citramalate synthase
- Curated proteins or TIGRFams with EC 2.3.1.182
- UniProt sequence Q8F3Q1_LEPIN: RecName: Full=(R)-citramalate synthase CimA {ECO:0000305}; EC=2.3.1.182 {ECO:0000269|PubMed:15292141, ECO:0000269|PubMed:18498255, ECO:0000269|PubMed:19351325}; AltName: Full=LiCMS {ECO:0000303|PubMed:18498255};
- UniProt sequence D4GSQ2: SubName: Full=2-isopropylmalate synthase / (R)-citramalatesynthase {ECO:0000313|EMBL:ADE04954.1}; EC=2.3.1.182 {ECO:0000313|EMBL:ADE04954.1}; EC=2.3.3.13 {ECO:0000313|EMBL:ADE04954.1};
- Comment: MetaCyc L-isoleucine biosynthesis II describes the formation of 2-oxobutanoate via citramalate. The other steps are the same (although it gives a different EC number for ilvC because of different cofactor preference) The citramalate synthase from Leptopsira interrogans (LA_2350, NP_712531, or Q8F3Q1_LEPIN) has been characterized biochemically but is not in the curated databases, see PMID:18498255 The putative citramalate synthase HVO_0644 (D4GSQ2) from Haloferax volcanii is required for isoleucine biosynthesis, see PMC4300041
- Total: 1 HMMs and 5 characterized proteins
leuC: citramalate isomerase large subunit
- Curated proteins matching citramalate isomerase large subunit
- Curated proteins matching 3-isopropylmalate dehydratase large subunit
- Curated proteins matching 3-isopropylmalate dehydratase%LeuC
- HMM TIGR00170
- HMM TIGR02083
- HMM TIGR02086
- Ignore hits to Q0QLE2 when looking for 'other' hits (2,3-dimethylmalate dehydratase large subunit; EC 4.2.1.85)
- Ignore hits to items matching EC 4.2.1.33 when looking for 'other' hits
- Ignore hits to items matching EC 4.2.1.35 when looking for 'other' hits
- UniProt sequence LEUC_DESVH: RecName: Full=3-isopropylmalate dehydratase large subunit {ECO:0000255|HAMAP-Rule:MF_01027}; EC=4.2.1.33 {ECO:0000255|HAMAP-Rule:MF_01027}; AltName: Full=Alpha-IPM isomerase {ECO:0000255|HAMAP-Rule:MF_01027}; Short=IPMI {ECO:0000255|HAMAP-Rule:MF_01027}; AltName: Full=Isopropylmalate isomerase {ECO:0000255|HAMAP-Rule:MF_01027};
- Ignore hits to CH_122621 when looking for 'other' hits (alpha isopropylmalate isomerase (Eurofung))
- Comment: In leucine synthesis, LeuCD allows the dehydration of 2-isopropylmalate and hydration to 3-isopropylmalate. Similarly, many of these enzymes allow the isomerization of citramalate to 3-methylmalate via citraconate. Citramalate isomerase is usually given as EC 4.2.1.35, as opposed to 4.2.1.33 for traditional leuCD. However, in initial testing, all of the bacteria with the citramalate pathway appeared to have "typical" leuBCD (Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, Bacteroides thetaiotaomicron, Magnetospirillum magneticum AMB-1, and Synechococcus elongatus PCC 7942). Ignore a 2,3-methylmalate dehydratase (Q0QLE2,Q0QLE1) which is >50% identical to leuCD from DvH (DVU2982,DVU2983) Ignore some BRENDA annotations without subunit information, and ignore CharProtDB::CH_122621 (leuCD fusion) which is not actually characterized DvH leuC (DVU2982) has similarity to both LeuC and to homoaconitase, and fitness data confirms its role in amino acid biosynthesis, so explicitly include it
- Total: 3 HMMs and 8 characterized proteins
leuD: citramalate isomerase small subunit
leuB: 3-methylmalate dehydrogenase
- Curated proteins or TIGRFams with EC 1.1.1.85
- Curated proteins or TIGRFams with EC 1.1.1.n5
- Comment: The dehydrogenase is encoded by a leuB-type enzyme. Similarly as for leuCD, any 3-isopropylmalate dehydrogenase should be assumed to be capable of this reaction
- Total: 1 HMMs and 43 characterized proteins
prpE: propionyl-CoA synthetase
ofoa: 2-oxobutanoate:ferredoxin oxidoreductase, alpha subunit
- UniProt sequence OFOA1_SULTO: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 1, subunit alpha {ECO:0000303|PubMed:27619895}; Short=OFOR1 {ECO:0000303|PubMed:27619895}; EC=1.2.7.11 {ECO:0000269|PubMed:27619895, ECO:0000305|PubMed:19027887};
- UniProt sequence OFOA_SULSP: RecName: Full=2-oxoacid:ferredoxin oxidoreductase subunit alpha {ECO:0000303|PubMed:8902625}; Short=OFOR {ECO:0000303|PubMed:11683888}; EC=1.2.7.11 {ECO:0000269|PubMed:11683888, ECO:0000269|PubMed:12009405, ECO:0000269|PubMed:8902625};
- UniProt sequence OFOA_SACSO: RecName: Full=2-oxoacid:ferredoxin oxidoreductase subunit alpha {ECO:0000303|PubMed:16466637}; Short=OFOR {ECO:0000305}; EC=1.2.7.11 {ECO:0000269|PubMed:16466637};
- UniProt sequence OFOA2_SULTO: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 2, subunit alpha {ECO:0000303|PubMed:27619895}; Short=OFOR2 {ECO:0000303|PubMed:27619895}; EC=1.2.7.11 {ECO:0000269|PubMed:27619895};
- UniProt sequence OFOA1_AERPE: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 1, subunit alpha {ECO:0000303|PubMed:15848165}; Short=OFOR1 {ECO:0000303|PubMed:15848165}; EC=1.2.7.11 {ECO:0000269|PubMed:15848165};
- UniProt sequence OFOA2_AERPE: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 2, subunit alpha {ECO:0000303|PubMed:15848165}; Short=OFOR2 {ECO:0000303|PubMed:15848165}; EC=1.2.7.11 {ECO:0000269|PubMed:15848165};
- Comment: The key reaction is alpha-ketobutyrate synthase or 2-oxobutanoate:ferredoxin oxidoreductase (in reverse) These are heterodimeric enzymes and the only one mentioned by MetaCyc is an enzyme from Sulfolobus tokodaii 7 that includes ST2300 (alpha subunit, OFOA_SULSP). The beta subunit is OFOB_SULSP (but metacyc seems not to know this). Some related enzymes also are believed to do this
- Total: 6 characterized proteins
ofob: 2-oxobutanoate:ferredoxin oxidoreductase, beta subunit
- UniProt sequence OFOB1_SULTO: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 1, subunit beta {ECO:0000303|PubMed:27619895}; Short=OFOR1 {ECO:0000303|PubMed:27619895}; EC=1.2.7.11 {ECO:0000269|PubMed:27619895, ECO:0000305|PubMed:19027887};
- UniProt sequence OFOB_SULSP: RecName: Full=2-oxoacid:ferredoxin oxidoreductase subunit beta {ECO:0000303|PubMed:8902625}; Short=OFOR {ECO:0000303|PubMed:11683888}; EC=1.2.7.11 {ECO:0000269|PubMed:11683888, ECO:0000269|PubMed:12009405, ECO:0000269|PubMed:8902625};
- UniProt sequence OFOB_SACSO: RecName: Full=2-oxoacid:ferredoxin oxidoreductase subunit beta {ECO:0000303|PubMed:16466637}; Short=OFOR {ECO:0000305}; EC=1.2.7.11 {ECO:0000269|PubMed:16466637};
- UniProt sequence OFOB2_SULTO: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 2, subunit beta {ECO:0000303|PubMed:27619895}; Short=OFOR2 {ECO:0000303|PubMed:27619895}; EC=1.2.7.11 {ECO:0000269|PubMed:27619895};
- UniProt sequence OFOB1_AERPE: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 1, subunit beta {ECO:0000303|PubMed:15848165}; Short=OFOR1 {ECO:0000303|PubMed:15848165}; EC=1.2.7.11 {ECO:0000269|PubMed:15848165};
- UniProt sequence OFOB2_AERPE: RecName: Full=2-oxoacid:ferredoxin oxidoreductase 2, subunit beta {ECO:0000303|PubMed:15848165}; Short=OFOR2 {ECO:0000303|PubMed:15848165}; EC=1.2.7.11 {ECO:0000269|PubMed:15848165};
- 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 the paper from 2019 on GapMind for amino acid biosynthesis, the paper from 2022 on GapMind for carbon sources, or view the source code, or see changes to Amino acid biosynthesis since the publication.
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