Definition of L-isoleucine biosynthesis

GapMind for Amino acid biosynthesis

Definition of L-isoleucine biosynthesis

# Isoleucine biosynthesis in GapMind is based on MetaCyc pathways
# L-isoleucine biosynthesis I (from threonine) (metacyc:ILEUSYN-PWY),
# II via citramalate (metacyc:PWY-5101),
# or IV from propanoate (metacyc:PWY-5104).
# 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 (metacyc:PWY-5103), 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.
# MetaCyc L-isoleucine biosynthesis V describes biosynthesis from 2-methylbutanoate, which
# is a fermentation end product in the rumen; this is an an unusual precursor
# so we did not include it.

# (Ignore some CharProtDB annotations with threonine deaminase but no EC).
# uniprot:B1N2N4 is included because it is active on both serine and threonine (PMID:19931317).
# uniprot:P09367 is ignored because it may be active on threonine as well as serine.
ilvA	threonine deaminase	EC:4.3.1.19	ignore_other:threonine deaminase	curated:BRENDA::B1N2N4	ignore:BRENDA::P09367

import val.steps:ilvI ilvH ilvC ilvD

# Q8NS92 is ignored because it is primarily a transcriptional regulator.
# Similarity to aromatic amino acid transaminases or tyrosine transaminases is ignored as they
# often are often non-specific.
ilvE	isoleucine transaminase	EC:2.6.1.42	ignore:SwissProt::Q8NS92	ignore_other:2.6.1.57	ignore_other:2.6.1.5

# 2-oxobutanoate is formed by deaminating threonine (pathway I, ilvA), via citramalate synthase (pathway II, cimA), or via propionyl-CoA (pathway III, prpE)
oxobutanoate: ilvA

# 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) 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.
cimA	(R)-citramalate synthase	EC:2.3.1.182	EC:2.3.3.21	uniprot:Q8F3Q1	uniprot:D4GSQ2

# 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 (uniprot:A0A1X9Z7T5) from Pedobacter sp. GW460-11-11-14-LB5 is important for fitness
# unless amino acids are added.
# uniprot:S3E7P8 is annotated as this in SwissProt but we did not find experimental evidence, so it is ignored.
leuC	3-isopropylmalate dehydratase / citramalate isomerase, large subunit	term:citramalate isomerase large subunit	term:3-isopropylmalate dehydratase large subunit	term:3-isopropylmalate dehydratase%LeuC	hmm:TIGR00170	hmm:TIGR02083	hmm:TIGR02086	ignore:SwissProt::Q0QLE2	ignore_other:EC 4.2.1.33	ignore_other:EC 4.2.1.35	uniprot:LEUC_DESVH	ignore:CharProtDB::CH_122621	ignore:SwissProt::S3E7P8	uniprot:A0A1X9Z7T5

# A mutant of BAC65258.1 (uniprot:Q845W4) was shown to be a leucine auxotroph in PMC155387.
# CA265_RS15840 (uniprot:A0A1X9Z766) from Pedobacter sp. GW460-11-11-14-LB5 is important for fitness
# unless amino acids are added.
leuD	3-isopropylmalate dehydaratase / citramalate isomerase, small subunit	term:citramalate isomerase small subunit	term:3-isopropylmalate dehydratase small subunit	term:3-isopropylmalate dehydratase%LeuD	hmm:TIGR00171	hmm:TIGR02084	hmm:TIGR02087	ignore:SwissProt::Q0QLE1	ignore_other:EC 4.2.1.33	ignore_other:EC 4.2.1.35	ignore:CharProtDB::CH_122621	uniprot:Q845W4	uniprot:A0A1X9Z766

# The short protein uniprot:P87267 is misannotated as this in BRENDA, so it is ignored.
leuB	3-methylmalate dehydrogenase / 3-isopropylmalate dehydrogenase	EC:1.1.1.85	ignore:BRENDA::P87267

oxobutanoate: cimA leuC leuD leuB

# uniprot:Q8ZKF6 is very similar to E. coli acs and likely has this activity, so it is ignored.
prpE	propionyl-CoA synthetase	term:propionyl-CoA synthetase	term:propionate--CoA ligase	EC:6.2.1.17	ignore:SwissProt::Q8ZKF6

# alpha-ketobutyrate synthase or
# 2-oxobutanoate:ferredoxin oxidoreductase (in reverse) is a heterodimeric enzyme
ofoa	2-oxobutanoate:ferredoxin oxidoreductase, alpha subunit	uniprot:OFOA1_SULTO	uniprot:OFOA_SULSP	uniprot:OFOA_SACSO	uniprot:OFOA2_SULTO	uniprot:OFOA1_AERPE	uniprot:OFOA2_AERPE	term:2-oxoacid:ferredoxin oxidoreductase%subunit alpha	curated:BRENDA::Q4J6I9

ofob	2-oxobutanoate:ferredoxin oxidoreductase, beta subunit	uniprot:OFOB1_SULTO	uniprot:OFOB_SULSP	uniprot:OFOB_SACSO	uniprot:OFOB2_SULTO	uniprot:OFOB1_AERPE	uniprot:OFOB2_AERPE	term:2-oxoacid:ferredoxin oxidoreductase%subunit beta	curated:BRENDA::Q4J6I8

oxobutanoate: prpE ofoa ofob

all: oxobutanoate ilvI ilvH ilvC ilvD ilvE

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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
the source code
instructions for running GapMind on your computer
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

GapMind for Amino acid biosynthesis