CIMA_LEPIN
ID CIMA_LEPIN Reviewed; 516 AA.
AC Q8F3Q1;
DT 22-APR-2020, integrated into UniProtKB/Swiss-Prot.
DT 01-MAR-2003, sequence version 1.
DT 03-AUG-2022, entry version 131.
DE RecName: Full=(R)-citramalate synthase CimA {ECO:0000305};
DE EC=2.3.1.182 {ECO:0000269|PubMed:15292141, ECO:0000269|PubMed:18498255, ECO:0000269|PubMed:19351325};
DE AltName: Full=LiCMS {ECO:0000303|PubMed:18498255};
GN Name=cimA {ECO:0000303|PubMed:15292141};
GN OrderedLocusNames=LA_2350 {ECO:0000312|EMBL:AAN49549.1};
OS Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai (strain
OS 56601).
OC Bacteria; Spirochaetes; Leptospirales; Leptospiraceae; Leptospira.
OX NCBI_TaxID=189518;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=56601;
RX PubMed=12712204; DOI=10.1038/nature01597;
RA Ren S.-X., Fu G., Jiang X.-G., Zeng R., Miao Y.-G., Xu H., Zhang Y.-X.,
RA Xiong H., Lu G., Lu L.-F., Jiang H.-Q., Jia J., Tu Y.-F., Jiang J.-X.,
RA Gu W.-Y., Zhang Y.-Q., Cai Z., Sheng H.-H., Yin H.-F., Zhang Y., Zhu G.-F.,
RA Wan M., Huang H.-L., Qian Z., Wang S.-Y., Ma W., Yao Z.-J., Shen Y.,
RA Qiang B.-Q., Xia Q.-C., Guo X.-K., Danchin A., Saint Girons I.,
RA Somerville R.L., Wen Y.-M., Shi M.-H., Chen Z., Xu J.-G., Zhao G.-P.;
RT "Unique physiological and pathogenic features of Leptospira interrogans
RT revealed by whole-genome sequencing.";
RL Nature 422:888-893(2003).
RN [2]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, PATHWAY, AND
RP INDUCTION.
RC STRAIN=56601;
RX PubMed=15292141; DOI=10.1128/jb.186.16.5400-5409.2004;
RA Xu H., Zhang Y., Guo X., Ren S., Staempfli A.A., Chiao J., Jiang W.,
RA Zhao G.;
RT "Isoleucine biosynthesis in Leptospira interrogans serotype lai strain
RT 56601 proceeds via a threonine-independent pathway.";
RL J. Bacteriol. 186:5400-5409(2004).
RN [3] {ECO:0007744|PDB:3BLE, ECO:0007744|PDB:3BLF, ECO:0007744|PDB:3BLI}
RP X-RAY CRYSTALLOGRAPHY (2.00 ANGSTROMS) OF 1-325 IN COMPLEXES WITH PYRUVATE;
RP ACETYL-COA; MALONATE AND ZINC, FUNCTION, CATALYTIC ACTIVITY, COFACTOR,
RP BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, DOMAIN, ACTIVE SITE, AND
RP MUTAGENESIS OF ARG-16; ASP-17; LEU-81; PHE-83; LEU-104; TYR-144; GLU-146;
RP THR-179; HIS-302; ASP-304; ASN-310; LEU-311 AND TYR-312.
RC STRAIN=56601;
RX PubMed=18498255; DOI=10.1042/bj20080242;
RA Ma J., Zhang P., Zhang Z., Zha M., Xu H., Zhao G., Ding J.;
RT "Molecular basis of the substrate specificity and the catalytic mechanism
RT of citramalate synthase from Leptospira interrogans.";
RL Biochem. J. 415:45-56(2008).
RN [4] {ECO:0007744|PDB:3F6G, ECO:0007744|PDB:3F6H}
RP X-RAY CRYSTALLOGRAPHY (2.00 ANGSTROMS) OF 390-516 IN COMPLEXES WITH ZINC
RP AND ISOLEUCINE, FUNCTION, CATALYTIC ACTIVITY, ACTIVITY REGULATION, SUBUNIT,
RP DOMAIN, AND MUTAGENESIS OF TYR-430; ASP-431; LEU-451; TYR-454; ILE-458;
RP THR-464; VAL-468; PRO-493 AND GLN-495.
RX PubMed=19351325; DOI=10.1042/bj20090336;
RA Zhang P., Ma J., Zhang Z., Zha M., Xu H., Zhao G., Ding J.;
RT "Molecular basis of the inhibitor selectivity and insights into the
RT feedback inhibition mechanism of citramalate synthase from Leptospira
RT interrogans.";
RL Biochem. J. 421:133-143(2009).
CC -!- FUNCTION: Catalyzes the condensation of pyruvate and acetyl-coenzyme A
CC to form (R)-citramalate (PubMed:15292141, PubMed:18498255,
CC PubMed:19351325). Shows strict substrate specificity for pyruvate.
CC Cannot use alpha-ketoisovalerate, alpha-ketobutyrate, alpha-
CC ketoisocaproate, alpha-ketoglutarate or glyoxylate (PubMed:15292141,
CC PubMed:18498255). {ECO:0000269|PubMed:15292141,
CC ECO:0000269|PubMed:18498255, ECO:0000269|PubMed:19351325}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=acetyl-CoA + H2O + pyruvate = (3R)-citramalate + CoA + H(+);
CC Xref=Rhea:RHEA:19045, ChEBI:CHEBI:15361, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:30934, ChEBI:CHEBI:57287,
CC ChEBI:CHEBI:57288; EC=2.3.1.182;
CC Evidence={ECO:0000269|PubMed:15292141, ECO:0000269|PubMed:18498255,
CC ECO:0000269|PubMed:19351325};
CC PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:19046;
CC Evidence={ECO:0000269|PubMed:15292141, ECO:0000269|PubMed:18498255,
CC ECO:0000269|PubMed:19351325};
CC -!- COFACTOR:
CC Name=Mn(2+); Xref=ChEBI:CHEBI:29035;
CC Evidence={ECO:0000269|PubMed:18498255};
CC Note=Mn(2+) is the most effective activator, followed by Co(2+),
CC Ca(2+), Mg(2+) and Ni(2+). {ECO:0000269|PubMed:18498255};
CC -!- ACTIVITY REGULATION: Regulated by the end-product isoleucine via a
CC feedback inhibition. The binding of isoleucine has inhibitory effects
CC on the binding of both pyruvate and acetyl-CoA. May act via
CC conformational change of the dimer interface of the regulatory domain,
CC leading to inhibition of the catalytic reaction.
CC {ECO:0000269|PubMed:19351325}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=43 uM for pyruvate {ECO:0000269|PubMed:15292141};
CC KM=60 uM for pyruvate {ECO:0000269|PubMed:18498255};
CC KM=1118 uM for acetyl-CoA {ECO:0000269|PubMed:18498255};
CC Note=kcat is 2.41 sec(-1) (PubMed:15292141). kcat is 10.3 sec(-1)
CC with acetyl-CoA as substrate (PubMed:18498255). kcat is 9.13 sec(-1)
CC with pyruvate as substrate (PubMed:18498255).
CC {ECO:0000269|PubMed:15292141, ECO:0000269|PubMed:18498255};
CC Temperature dependence:
CC Optimum temperature is 35-40 degrees Celsius.
CC {ECO:0000269|PubMed:15292141};
CC -!- PATHWAY: Amino-acid biosynthesis; L-isoleucine biosynthesis; 2-
CC oxobutanoate from pyruvate: step 1/3. {ECO:0000269|PubMed:15292141}.
CC -!- SUBUNIT: Homodimer. {ECO:0000269|PubMed:18498255,
CC ECO:0000269|PubMed:19351325}.
CC -!- INDUCTION: Expression is repressed by isoleucine but not by leucine.
CC {ECO:0000269|PubMed:15292141}.
CC -!- DOMAIN: Contains a catalytic N-terminal domain and a C-terminal
CC regulatory domain, linked together by a flexible region
CC (PubMed:18498255, PubMed:19351325). The catalytic domain consists of a
CC TIM barrel flanked by an extended C-terminal region. The active site is
CC located at the center of the TIM barrel near the C-terminal ends of the
CC beta-strands and is composed of conserved residues of the beta-strands
CC of one subunit and the C-terminal region of the other
CC (PubMed:18498255). {ECO:0000269|PubMed:18498255,
CC ECO:0000269|PubMed:19351325}.
CC -!- SIMILARITY: Belongs to the alpha-IPM synthase/homocitrate synthase
CC family. {ECO:0000305}.
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DR EMBL; AE010300; AAN49549.1; -; Genomic_DNA.
DR RefSeq; NP_712531.1; NC_004342.2.
DR RefSeq; WP_000169689.1; NC_004342.2.
DR PDB; 3BLE; X-ray; 2.00 A; A=1-325.
DR PDB; 3BLF; X-ray; 2.60 A; A=1-325.
DR PDB; 3BLI; X-ray; 2.50 A; A=1-325.
DR PDB; 3F6G; X-ray; 2.00 A; A/B=390-516.
DR PDB; 3F6H; X-ray; 2.70 A; A/B=390-516.
DR PDBsum; 3BLE; -.
DR PDBsum; 3BLF; -.
DR PDBsum; 3BLI; -.
DR PDBsum; 3F6G; -.
DR PDBsum; 3F6H; -.
DR AlphaFoldDB; Q8F3Q1; -.
DR SMR; Q8F3Q1; -.
DR STRING; 189518.LA_2350; -.
DR EnsemblBacteria; AAN49549; AAN49549; LA_2350.
DR KEGG; lil:LA_2350; -.
DR PATRIC; fig|189518.3.peg.2333; -.
DR HOGENOM; CLU_022158_0_1_12; -.
DR OMA; DWSNGMR; -.
DR BioCyc; MetaCyc:MON-11894; -.
DR UniPathway; UPA00047; UER00066.
DR EvolutionaryTrace; Q8F3Q1; -.
DR Proteomes; UP000001408; Chromosome I.
DR GO; GO:0043714; F:(R)-citramalate synthase activity; IEA:UniProtKB-EC.
DR GO; GO:0003852; F:2-isopropylmalate synthase activity; IBA:GO_Central.
DR GO; GO:0016829; F:lyase activity; IEA:UniProtKB-KW.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0009097; P:isoleucine biosynthetic process; IEA:UniProtKB-UniPathway.
DR GO; GO:0009098; P:leucine biosynthetic process; IBA:GO_Central.
DR Gene3D; 3.20.20.70; -; 1.
DR InterPro; IPR013709; 2-isopropylmalate_synth_dimer.
DR InterPro; IPR002034; AIPM/Hcit_synth_CS.
DR InterPro; IPR013785; Aldolase_TIM.
DR InterPro; IPR036230; LeuA_allosteric_dom_sf.
DR InterPro; IPR000891; PYR_CT.
DR Pfam; PF00682; HMGL-like; 1.
DR Pfam; PF08502; LeuA_dimer; 1.
DR SMART; SM00917; LeuA_dimer; 1.
DR SUPFAM; SSF110921; SSF110921; 1.
DR PROSITE; PS00815; AIPM_HOMOCIT_SYNTH_1; 1.
DR PROSITE; PS50991; PYR_CT; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Amino-acid biosynthesis;
KW Branched-chain amino acid biosynthesis; Isoleucine biosynthesis; Lyase;
KW Manganese; Metal-binding; Pyruvate; Reference proteome; Transferase.
FT CHAIN 1..516
FT /note="(R)-citramalate synthase CimA"
FT /id="PRO_0000449424"
FT DOMAIN 8..269
FT /note="Pyruvate carboxyltransferase"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01151"
FT ACT_SITE 16
FT /note="Proton donor"
FT /evidence="ECO:0000305|PubMed:18498255"
FT ACT_SITE 146
FT /note="Proton acceptor"
FT /evidence="ECO:0000305|PubMed:18498255"
FT BINDING 16..17
FT /ligand="pyruvate"
FT /ligand_id="ChEBI:CHEBI:15361"
FT /evidence="ECO:0000269|PubMed:18498255"
FT BINDING 17
FT /ligand="Mn(2+)"
FT /ligand_id="ChEBI:CHEBI:29035"
FT /evidence="ECO:0000305|PubMed:18498255,
FT ECO:0000305|PubMed:19351325"
FT BINDING 144
FT /ligand="pyruvate"
FT /ligand_id="ChEBI:CHEBI:15361"
FT /evidence="ECO:0000269|PubMed:18498255"
FT BINDING 179
FT /ligand="pyruvate"
FT /ligand_id="ChEBI:CHEBI:15361"
FT /evidence="ECO:0000269|PubMed:18498255"
FT BINDING 207
FT /ligand="Mn(2+)"
FT /ligand_id="ChEBI:CHEBI:29035"
FT /evidence="ECO:0000305|PubMed:18498255,
FT ECO:0000305|PubMed:19351325"
FT BINDING 209
FT /ligand="Mn(2+)"
FT /ligand_id="ChEBI:CHEBI:29035"
FT /evidence="ECO:0000305|PubMed:18498255,
FT ECO:0000305|PubMed:19351325"
FT MUTAGEN 16
FT /note="R->K,Q: Loss of activity."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 17
FT /note="D->A: 34-fold increase in Km for pyruvate and 315-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 17
FT /note="D->N: 4.4-fold increase in Km for pyruvate and 480-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 81
FT /note="L->A: 4.7-fold increase in Km for pyruvate and 15.7-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 81
FT /note="L->V: 3.3-fold increase in Km for pyruvate and 10.1-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 83
FT /note="F->A: 5-fold increase in Km for acetyl-CoA and 120-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 104
FT /note="L->V: 1.8-fold increase in Km for pyruvate and 3.4-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 144
FT /note="Y->L: 259-fold increase in Km for pyruvate and 76-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 144
FT /note="Y->V: 114-fold increase in Km for pyruvate and 5.3-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 146
FT /note="E->D,Q: Minor effects on the binding of acetyl-CoA,
FT but causes a strong decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 179
FT /note="T->A: 16.4-fold increase in Km for pyruvate and 186-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 302
FT /note="H->A,N: Loss of activity."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 304
FT /note="D->A: 5.2-fold increase in Km for acetyl-CoA and
FT 16.6-fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 310
FT /note="N->A: 2.2-fold increase in Km for acetyl-CoA and
FT 1.7-fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 311
FT /note="L->A: 8-fold increase in Km for acetyl-CoA and 6-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 312
FT /note="Y->A: Loss of activity."
FT /evidence="ECO:0000269|PubMed:18498255"
FT MUTAGEN 430
FT /note="Y->L: No change in Km for acetyl-CoA and 2.3-fold
FT decrease in kcat. Severely impairs inhibition by
FT isoleucine."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 431
FT /note="D->A: 1.8-fold decrease in Km for acetyl-CoA and 5-
FT fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 451
FT /note="L->V: 1.5-fold increase in Km for acetyl-CoA and 4.3
FT decrease in kcat."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 454
FT /note="Y->A: 1.4 decrease in Km for acetyl-CoA and 17-fold
FT decrease in kcat. Still inhibited by isoleucine and weakly
FT inhibited by leucine."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 458
FT /note="I->A: 1.3-fold decrease in Km for acetyl-CoA and 14-
FT fold decrease in kcat. Abolishes inhibition by isoleucine."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 464
FT /note="T->A: 1.8-fold decrease in Km for acetyl-CoA and
FT 4.3-fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 468
FT /note="V->A: No change in Km for acetyl-CoA and 2-fold
FT decrease in kcat. Increases inhibition by isoleucine and
FT leucine becomes an effective inhibitor."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 493
FT /note="P->A: 1.5-fold decrease in Km for acetyl-CoA and
FT 2.6-fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:19351325"
FT MUTAGEN 495
FT /note="Q->A: 1.6-fold decrease in Km for acetyl-CoA and
FT 2.8-fold decrease in kcat."
FT /evidence="ECO:0000269|PubMed:19351325"
FT STRAND 9..12
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 14..18
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 28..40
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 45..51
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 58..71
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 75..77
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 78..85
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 88..96
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 100..106
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 109..115
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 120..136
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 140..146
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 148..154
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 156..167
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 172..177
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 185..198
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 204..207
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 215..224
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 228..233
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 234..236
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 238..241
FT /evidence="ECO:0007829|PDB:3BLI"
FT HELIX 246..256
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 265..267
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 268..279
FT /evidence="ECO:0007829|PDB:3BLE"
FT TURN 288..290
FT /evidence="ECO:0007829|PDB:3BLE"
FT TURN 292..295
FT /evidence="ECO:0007829|PDB:3BLE"
FT HELIX 318..321
FT /evidence="ECO:0007829|PDB:3BLE"
FT STRAND 392..401
FT /evidence="ECO:0007829|PDB:3F6G"
FT STRAND 408..415
FT /evidence="ECO:0007829|PDB:3F6G"
FT STRAND 418..425
FT /evidence="ECO:0007829|PDB:3F6G"
FT HELIX 429..444
FT /evidence="ECO:0007829|PDB:3F6G"
FT STRAND 450..457
FT /evidence="ECO:0007829|PDB:3F6G"
FT STRAND 468..475
FT /evidence="ECO:0007829|PDB:3F6G"
FT HELIX 482..484
FT /evidence="ECO:0007829|PDB:3F6G"
FT STRAND 486..494
FT /evidence="ECO:0007829|PDB:3F6G"
FT HELIX 495..510
FT /evidence="ECO:0007829|PDB:3F6G"
SQ SEQUENCE 516 AA; 57319 MW; 4F0DE93214537124 CRC64;
MTKVETRLEI LDVTLRDGEQ TRGVSFSTSE KLNIAKFLLQ KLNVDRVEIA SARVSKGELE
TVQKIMEWAA TEQLTERIEI LGFVDGNKTV DWIKDSGAKV LNLLTKGSLH HLEKQLGKTP
KEFFTDVSFV IEYAIKSGLK INVYLEDWSN GFRNSPDYVK SLVEHLSKEH IERIFLPDTL
GVLSPEETFQ GVDSLIQKYP DIHFEFHGHN DYDLSVANSL QAIRAGVKGL HASINGLGER
AGNTPLEALV TTIHDKSNSK TNINEIAITE ASRLVEVFSG KRISANRPIV GEDVFTQTAG
VHADGDKKGN LYANPILPER FGRKRSYALG KLAGKASISE NVKQLGMVLS EVVLQKVLER
VIELGDQNKL VTPEDLPFII ADVSGRTGEK VLTIKSCNIH SGIGIRPHAQ IELEYQGKIH
KEISEGDGGY DAFMNALTKI TNRLGISIPK LIDYEVRIPP GGKTDALVET RITWNKSLDL
EEDQTFKTMG VHPDQTVAAV HATEKMLNQI LQPWQI
