Definition of L-leucine biosynthesis
As rules and steps, or see full text
Rules
Overview: Leucine biosynthesis in GapMind is based on MetaCyc pathway L-leucine biosynthesis (link). This begins with the initial steps of valine biosynthesis (see link), up to 3-methyloxobutanoate.
Steps
ilvI: acetolactate/acetohydroxybutanoate synthase catalytic subunit
ilvH: acetolactate/acetohydroxybutanoate synthase regulatory subunit
- HMM TIGR00119
- Curated proteins matching acetohydroxy-acid synthase%small
- Curated proteins matching acetohydroxybutanoate synthase, regulatory subunit
- Curated proteins matching small subunit of acetolactate synthase
- Curated sequence P0ADG1: acetolactate synthase (subunit 1/2) (EC 2.2.1.6). acetolactate synthase II subunit IlvM (EC 2.2.1.6)
- Ignore hits to items matching EC 2.2.1.6 when looking for 'other' hits
- UniProt sequence A0A154R0Y7: RecName: Full=Acetolactate synthase small subunit {ECO:0000256|RuleBase:RU368092}; Short=AHAS {ECO:0000256|RuleBase:RU368092}; Short=ALS {ECO:0000256|RuleBase:RU368092}; EC=2.2.1.6 {ECO:0000256|RuleBase:RU368092}; AltName: Full=Acetohydroxy-acid synthase small subunit {ECO:0000256|RuleBase:RU368092};
- Curated sequence Q93YZ7: Acetolactate synthase small subunit 1, chloroplastic; ALS-interacting protein 1; Acetohydroxyacid synthase small subunit 1
- UniProt sequence A0A0H2X4P1: SubName: Full=Acetolactate synthase isozyme II small subunit {ECO:0000313|EMBL:AAY47914.1};
- Predicted: UniProt sequence A0A7J3AYJ4: SubName: Full=Uncharacterized protein {ECO:0000313|EMBL:HGD33185.1};
- Predicted: UniProt sequence A0A161J739: SubName: Full=Acetolactate synthase 3 regulatory subunit domain protein {ECO:0000313|EMBL:ANC53960.1};
- Comment: The isolated catalytic subunit can have some activity on its own, so it's not clear if the regulatory subunit (ilvH) is always required, but ilvH does always seem to be present. P0ADG1 is annotated with this EC number but not explicitly as the small regulatory subunit, so it was added manually. Q93YZ7 is annotated as this but without the EC number, so is added manually. Most regulatory subunits have an N-terminal ACT domain and a C-terminal ACT-like domain, but E. coli IlvM, which is required for the activity of E. coli acetohydroxyacid synthase isoenzyme II, has the N-terminal ACT domain only. We identified several other short (one-domain) regulatory subunits. In Rhodanobacter and related genera, the putative regulatory subunit has just one ACT domain (i.e., LRK54_RS10305, which is nearly identical to A0A154R0Y7). Based on sequence analysis, short ilvH probably maintains the ability to bind valine and to bind the catalytic subunit, but not the ability to bind ATP or other regulatory subunits. Mutant fitness data confirms that LRK54_RS10305 is involved in amino acid biosynthesis. In Xanthomonas campestris, the one ACT-domain protein Xcc-8004.1058.1 (A0A0H2X4P1), which is conserved next to ilvI, has a similar fitness pattern as ilvI (Alice Castaing, unpublished data). Furthermore, its AlphaFold structure is very similar to that of E. coli IlvM (TM-score 0.89, RMSD 1.49 A, foldseek). So Xcc-8004.1058.1 is another short regulatory subunit. Similarly, in Brevundimonas sp. GW460-12-10-14-LB2, the putative ilvH has the ACT domain only (Brev2_1981 = A0A161J739). Many Thermoproteota seem to have a diverged short regulatory subunit, such as the ACT domain protein KCR_RS03285 (A0A7J3AYJ4), which is conserved next to ilvI. Foldseek shows that this protein is similar to the ACT domain of (p)ppGpp synthase but also to IlvH of Staphylococcus aureus, so we predict that it is the regulatory subunit.
- Total: 1 HMMs and 12 characterized proteins
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
- Curated sequence CH_123630: mitochondrial ketol-acid reductoisomerase
- 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. CH_123630 is added because it is annotated as this but with no EC number.
- Total: 1 HMMs and 34 characterized proteins
ilvD: dihydroxy-acid 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: IlvD is involved in the biosynthesis of isoleucine, with (R)-2,3-dihydroxy-3-methylpentanoate as the substrate, and in the biosynthesis of valine and leucine, with (R)-2,3-dihydroxy-3-methylbutanoate as the substrate. 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 12 characterized proteins
leuB: 3-methylmalate dehydrogenase / 3-isopropylmalate dehydrogenase
leuC: 3-isopropylmalate dehydratase / 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. dimethylmaleate hydratase subunit A (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))
- Ignore hits to S3E7P8 when looking for 'other' hits (3-isopropylmalate dehydratase large subunit gloJ; L-homotyrosine biosynthesis sub-cluster protein gloJ; Pneumocandin biosynthesis cluster protein J; EC 4.2.1.33)
- UniProt sequence A0A1X9Z7T5: RecName: Full=3-isopropylmalate dehydratase large subunit {ECO:0000256|HAMAP-Rule:MF_01026}; EC=4.2.1.33 {ECO:0000256|HAMAP-Rule:MF_01026}; AltName: Full=Alpha-IPM isomerase {ECO:0000256|HAMAP-Rule:MF_01026}; Short=IPMI {ECO:0000256|HAMAP-Rule:MF_01026}; AltName: Full=Isopropylmalate isomerase {ECO:0000256|HAMAP-Rule:MF_01026};
- 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, many the bacteria with the citramalate pathway appeared to have "typical" leuBCD (i.e., Desulfovibrio vulgaris Hildenborough, Desulfovibrio vulgaris Miyazaki F, Bacteroides thetaiotaomicron, Magnetospirillum magneticum AMB-1, and Synechococcus elongatus PCC 7942). So we do not try to distinguish between 3-isoprpylmalate dehydratase and citramalate isomerase. 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. CA265_RS15830 (A0A1X9Z7T5) from Pedobacter sp. GW460-11-11-14-LB5 is important for fitness unless amino acids are added. S3E7P8 is annotated as this in SwissProt but we did not find experimental evidence, so it is ignored.
- Total: 3 HMMs and 9 characterized proteins
leuD: 3-isopropylmalate dehydaratase / citramalate isomerase, small subunit
- Curated proteins matching citramalate isomerase small subunit
- Curated proteins matching 3-isopropylmalate dehydratase small subunit
- Curated proteins matching 3-isopropylmalate dehydratase%LeuD
- HMM TIGR00171
- HMM TIGR02084
- HMM TIGR02087
- Ignore hits to Q0QLE1 when looking for 'other' hits (2,3-dimethylmalate dehydratase small subunit; EC 4.2.1.85. dimethylmaleate hydratase subunit B (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
- Ignore hits to CH_122621 when looking for 'other' hits (alpha isopropylmalate isomerase (Eurofung))
- UniProt sequence Q845W4: RecName: Full=3-isopropylmalate dehydratase small subunit {ECO:0000256|HAMAP-Rule:MF_01031}; EC=4.2.1.33 {ECO:0000256|HAMAP-Rule:MF_01031}; AltName: Full=Alpha-IPM isomerase {ECO:0000256|HAMAP-Rule:MF_01031}; Short=IPMI {ECO:0000256|HAMAP-Rule:MF_01031}; AltName: Full=Isopropylmalate isomerase {ECO:0000256|HAMAP-Rule:MF_01031};
- UniProt sequence A0A1X9Z766: RecName: Full=3-isopropylmalate dehydratase small subunit {ECO:0000256|HAMAP-Rule:MF_01031}; EC=4.2.1.33 {ECO:0000256|HAMAP-Rule:MF_01031}; AltName: Full=Alpha-IPM isomerase {ECO:0000256|HAMAP-Rule:MF_01031}; Short=IPMI {ECO:0000256|HAMAP-Rule:MF_01031}; AltName: Full=Isopropylmalate isomerase {ECO:0000256|HAMAP-Rule:MF_01031};
- Comment: A mutant of BAC65258.1 (Q845W4) was shown to be a leucine auxotroph in PMC155387. CA265_RS15840 (A0A1X9Z766) from Pedobacter sp. GW460-11-11-14-LB5 is important for fitness unless amino acids are added.
- Total: 3 HMMs and 11 characterized proteins
leuA: 2-isopropylmalate synthase
- Curated proteins or TIGRFams with EC 2.3.3.13
- UniProt sequence L0G5E1_ECHVK: SubName: Full=Isopropylmalate/homocitrate/citramalate synthase {ECO:0000313|EMBL:AGA80045.1};
- UniProt sequence A0A1X9Z750_9SPHI: SubName: Full=2-isopropylmalate synthase {ECO:0000313|EMBL:ARS41048.1};
- Ignore hits to items matching isopropylmalate synthase when looking for 'other' hits
- Ignore hits to P85362 when looking for 'other' hits (2-isopropylmalate synthase; Alpha-IPM synthase; Alpha-isopropylmalate synthase; EC 2.3.3.13)
- Ignore hits to S3D9F8 when looking for 'other' hits (Isopropyl malate synthase gloH; L-homotyrosine biosynthesis sub-cluster protein gloH; Pneumocandin biosynthesis cluster protein H; EC 2.3.3.13)
- Ignore hits to K0E4E5 when looking for 'other' hits (Isopropyl malate synthase htyA; L-homotyrosine biosynthetic cluster protein A; EC 2.3.3.13. 2-(4-hydroxybenzyl)-malate synthase)
- Comment: Ignore CharProtDB entries with no EC number. Add Echvi_3833 (L0G5E1_ECHVK) and CA265_RS15855 (A0A1X9Z750_9SPHI), which are supported by fitness data. P85362 is ignored because it is just 9 amino acids. S3D9F8 and K0E4E5 are involved in secondary metabolite synthesis and it is not clear if they actually have this activity, so they are ignored.
- Total: 2 HMMs and 30 characterized proteins
ilvE: leucine transaminase
- Curated proteins or TIGRFams with EC 2.6.1.6
- Curated proteins or TIGRFams with EC 2.6.1.42
- Ignore hits to Q8NS92 when looking for 'other' hits (HTH-type pyridoxine biosynthesis transcriptional regulator PdxR)
- Ignore hits to items matching 2.6.1.57 when looking for 'other' hits
- Ignore hits to items matching 2.6.1.5 when looking for 'other' hits
- Comment: Q8NS92 is ignored because it is primarily a transcriptional regulator. Similarity to aromatic amino acid transaminases or tyrosine transaminases is ignored as they are often non-specific.
- Total: 2 HMMs and 59 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