MAAL_CLOTT
ID MAAL_CLOTT Reviewed; 413 AA.
AC Q05514;
DT 01-FEB-1995, integrated into UniProtKB/Swiss-Prot.
DT 01-FEB-1995, sequence version 1.
DT 03-AUG-2022, entry version 105.
DE RecName: Full=Methylaspartate ammonia-lyase;
DE Short=MAL;
DE EC=4.3.1.2;
DE AltName: Full=3-methylaspartase ammonia-lyase;
DE AltName: Full=Beta-methylaspartase;
OS Clostridium tetanomorphum.
OC Bacteria; Firmicutes; Clostridia; Eubacteriales; Clostridiaceae;
OC Clostridium.
OX NCBI_TaxID=1553;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 1-26, FUNCTION,
RP CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, AND SUBUNIT.
RC STRAIN=ATCC 15920 / DSM 528 / NCIMB 11547 / H1;
RX PubMed=1420191; DOI=10.1021/bi00159a015;
RA Goda S.K., Minton N.P., Botting N.P., Gani D.;
RT "Cloning, sequencing, and expression in Escherichia coli of the Clostridium
RT tetanomorphum gene encoding beta-methylaspartase and characterization of
RT the recombinant protein.";
RL Biochemistry 31:10747-10756(1992).
RN [2]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-24, AND PROTEIN SEQUENCE OF 1-24.
RC STRAIN=ATCC 15920 / DSM 528 / NCIMB 11547 / H1;
RX PubMed=8454064; DOI=10.1016/0014-5793(93)80042-s;
RA Brecht M., Kellermann J., Plueckthun A.;
RT "Cloning and sequencing of glutamate mutase component E from Clostridium
RT tetanomorphum.";
RL FEBS Lett. 319:84-89(1993).
RN [3]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-71.
RC STRAIN=ATCC 15920 / DSM 528 / NCIMB 11547 / H1;
RX PubMed=8428631; DOI=10.1016/0014-5793(93)81488-l;
RA Holloway D.E., Marsh E.N.G.;
RT "Cloning and sequencing of glutamate mutase component E from Clostridium
RT tetanomorphum. Organization of the mut genes.";
RL FEBS Lett. 317:44-48(1993).
RN [4]
RP PROTEIN SEQUENCE OF 1-15.
RC STRAIN=ATCC 15920 / DSM 528 / NCIMB 11547 / H1;
RX PubMed=1397267; DOI=10.1016/0014-5793(92)81321-c;
RA Marsh E.N.G., Holloway D.E.;
RT "Cloning and sequencing of glutamate mutase component S from Clostridium
RT tetanomorphum. Homologies with other cobalamin-dependent enzymes.";
RL FEBS Lett. 310:167-170(1992).
RN [5]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, ACTIVITY
RP REGULATION, SUBSTRATE SPECIFICITY, AND COFACTOR.
RC STRAIN=ATCC 15920 / DSM 528 / NCIMB 11547 / H1;
RX PubMed=13630903; DOI=10.1016/s0021-9258(18)70297-4;
RA Barker H.A., Smyth R.D., Wilson R.M., Weissbach H.;
RT "The purification and properties of beta-methylaspartase.";
RL J. Biol. Chem. 234:320-328(1959).
RN [6]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF
RP HIS-194; GLN-329 AND LYS-331, SUBSTRATE SPECIFICITY, ACTIVE SITE, AND
RP SUBUNIT.
RC STRAIN=ATCC 15920 / DSM 528 / NCIMB 11547 / H1;
RX PubMed=19670200; DOI=10.1002/cbic.200900311;
RA Raj H., Weiner B., Veetil V.P., Reis C.R., Quax W.J., Janssen D.B.,
RA Feringa B.L., Poelarends G.J.;
RT "Alteration of the diastereoselectivity of 3-methylaspartate ammonia lyase
RT by using structure-based mutagenesis.";
RL ChemBioChem 10:2236-2245(2009).
RN [7]
RP X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) IN COMPLEX WITH MAGNESIUM, ACTIVE
RP SITE, COFACTOR, AND SUBUNIT.
RX PubMed=11748244; DOI=10.1074/jbc.m111180200;
RA Asuncion M., Blankenfeldt W., Barlow J.N., Gani D., Naismith J.H.;
RT "The structure of 3-methylaspartase from Clostridium tetanomorphum
RT functions via the common enolase chemical step.";
RL J. Biol. Chem. 277:8306-8311(2002).
RN [8]
RP X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) IN COMPLEX WITH SUBSTRATE ANALOGS
RP AND MAGNESIUM, FUNCTION, CATALYTIC ACTIVITY, MUTAGENESIS OF GLN-73 AND
RP LEU-384, COFACTOR, AND SUBUNIT.
RX PubMed=22614383; DOI=10.1038/nchem.1338;
RA Raj H., Szymanski W., de Villiers J., Rozeboom H.J., Veetil V.P.,
RA Reis C.R., de Villiers M., Dekker F.J., de Wildeman S., Quax W.J.,
RA Thunnissen A.M., Feringa B.L., Janssen D.B., Poelarends G.J.;
RT "Engineering methylaspartate ammonia lyase for the asymmetric synthesis of
RT unnatural amino acids.";
RL Nat. Chem. 4:478-484(2012).
CC -!- FUNCTION: Involved in the methylaspartate cycle. Catalyzes the
CC formation of the alpha,beta-unsaturated bond by the reversible anti
CC elimination of ammonia from L-threo-beta-methylaspartate (L-threo-
CC (2S,3S)-3-methylaspartate) to give mesaconate. It can also use L-
CC erythro-beta-methylaspartate (L-erythro-(2S,3R)-3-methylaspartate), L-
CC aspartate, fumarate and ethylfumarate as substrates.
CC {ECO:0000269|PubMed:13630903, ECO:0000269|PubMed:1420191,
CC ECO:0000269|PubMed:19670200, ECO:0000269|PubMed:22614383}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(2S,3S)-3-methyl-L-aspartate = mesaconate + NH4(+);
CC Xref=Rhea:RHEA:12829, ChEBI:CHEBI:28938, ChEBI:CHEBI:36986,
CC ChEBI:CHEBI:58724; EC=4.3.1.2; Evidence={ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200,
CC ECO:0000269|PubMed:22614383};
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000269|PubMed:11748244, ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:22614383};
CC -!- ACTIVITY REGULATION: Inhibited by calcium ions.
CC {ECO:0000269|PubMed:13630903}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.65 mM for L-threo-beta-methylaspartate (with 4 mM of KCl at pH
CC 9.76 and at 25 degrees Celsius) {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC KM=0.67 mM for L-threo-beta-methylaspartate (with 50 mM of KCl at pH
CC 9 and at 30 degrees Celsius) {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC KM=0.7 mM for mesaconate (with 20 mM of MgCl(2) at pH 9 and at 30
CC degrees Celsius) {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC KM=1 mM for L-threo-beta-methylaspartate (with 20 mM of MgCl(2) at pH
CC 9 and at 30 degrees Celsius) {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC KM=2.3 mM for L-aspartate (with 4 mM of KCl at pH 9.76 and at 25
CC degrees Celsius) {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC KM=2.8 mM for L-threo-beta-methylaspartate (with 0.3 mM of KCl at pH
CC 9 and at 30 degrees Celsius) {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC Vmax=2089 umol/min/mg enzyme with L-threo-beta-methylaspartate as
CC substrate (with 50 mM of KCl at pH 9 and at 30 degrees Celsius)
CC {ECO:0000269|PubMed:13630903, ECO:0000269|PubMed:1420191,
CC ECO:0000269|PubMed:19670200};
CC Vmax=309 umol/min/mg enzyme with L-threo-beta-methylaspartate as
CC substrate (with 0.3 mM of KCl at pH 9 and at 30 degrees Celsius)
CC {ECO:0000269|PubMed:13630903, ECO:0000269|PubMed:1420191,
CC ECO:0000269|PubMed:19670200};
CC Vmax=266 umol/min/mg enzyme with L-threo-beta-methylaspartate as
CC substrate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius)
CC {ECO:0000269|PubMed:13630903, ECO:0000269|PubMed:1420191,
CC ECO:0000269|PubMed:19670200};
CC Vmax=2.5 umol/min/mg enzyme with L-erythro-beta-methylaspartate as
CC substrate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius)
CC {ECO:0000269|PubMed:13630903, ECO:0000269|PubMed:1420191,
CC ECO:0000269|PubMed:19670200};
CC Vmax=2.4 umol/min/mg enzyme with L-aspartate as substrate (with 4 mM
CC of KCl at pH 9.76 and at 25 degrees Celsius)
CC {ECO:0000269|PubMed:13630903, ECO:0000269|PubMed:1420191,
CC ECO:0000269|PubMed:19670200};
CC Note=kcat is 61 sec(-1) for amination of mesaconate (with 20 mM of
CC MgCl(2) at pH 9 and at 30 degrees Celsius). kcat is 89 sec(-1) for
CC deamination of L-threo-beta-methylaspartate (with 20 mM of MgCl(2) at
CC pH 9 and at 30 degrees Celsius).;
CC pH dependence:
CC Optimum pH is 9.7. {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC Temperature dependence:
CC Optimum temperature is 55 degrees Celsius. It retains only half of
CC its original activity after a 30 minutes incubation period at 50
CC degrees Celsius. {ECO:0000269|PubMed:13630903,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200};
CC -!- PATHWAY: Amino-acid degradation; L-glutamate degradation via mesaconate
CC pathway; acetate and pyruvate from L-glutamate: step 2/4.
CC -!- SUBUNIT: Homodimer. {ECO:0000269|PubMed:11748244,
CC ECO:0000269|PubMed:1420191, ECO:0000269|PubMed:19670200,
CC ECO:0000269|PubMed:22614383}.
CC -!- SIMILARITY: Belongs to the methylaspartate ammonia-lyase family.
CC {ECO:0000305}.
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DR EMBL; S48141; AAB24070.1; -; Genomic_DNA.
DR EMBL; X70499; CAA49911.1; -; Genomic_DNA.
DR EMBL; X70695; CAA50027.1; -; Genomic_DNA.
DR PIR; B44285; B44285.
DR PDB; 1KCZ; X-ray; 1.90 A; A/B=1-413.
DR PDB; 1KD0; X-ray; 1.90 A; A/B=1-413.
DR PDB; 3ZVH; X-ray; 1.99 A; A/B=1-413.
DR PDB; 3ZVI; X-ray; 1.90 A; A/B=1-413.
DR PDBsum; 1KCZ; -.
DR PDBsum; 1KD0; -.
DR PDBsum; 3ZVH; -.
DR PDBsum; 3ZVI; -.
DR AlphaFoldDB; Q05514; -.
DR SMR; Q05514; -.
DR DrugBank; DB03661; L-cysteic acid.
DR BioCyc; MetaCyc:MON-1103; -.
DR BRENDA; 4.3.1.2; 1527.
DR UniPathway; UPA00561; UER00618.
DR EvolutionaryTrace; Q05514; -.
DR GO; GO:0031419; F:cobalamin binding; IEA:UniProtKB-KW.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0050096; F:methylaspartate ammonia-lyase activity; IEA:UniProtKB-EC.
DR GO; GO:0019553; P:glutamate catabolic process via L-citramalate; IEA:UniProtKB-UniPathway.
DR CDD; cd03314; MAL; 1.
DR Gene3D; 3.20.20.120; -; 1.
DR Gene3D; 3.30.390.10; -; 1.
DR InterPro; IPR036849; Enolase-like_C_sf.
DR InterPro; IPR029017; Enolase-like_N.
DR InterPro; IPR006395; Me_Asp_am_lyase.
DR InterPro; IPR022662; MeAsp_NH4-lyase_C.
DR InterPro; IPR022665; MeAsp_NH4-lyase_N.
DR Pfam; PF07476; MAAL_C; 1.
DR Pfam; PF05034; MAAL_N; 1.
DR PIRSF; PIRSF017107; MAL; 1.
DR SFLD; SFLDF00007; methylaspartate_ammonia-lyase; 1.
DR SUPFAM; SSF51604; SSF51604; 1.
DR SUPFAM; SSF54826; SSF54826; 1.
DR TIGRFAMs; TIGR01502; B_methylAsp_ase; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Cobalamin; Cobalt; Direct protein sequencing; Lyase;
KW Magnesium; Metal-binding.
FT CHAIN 1..413
FT /note="Methylaspartate ammonia-lyase"
FT /id="PRO_0000084547"
FT ACT_SITE 331
FT /note="Proton acceptor"
FT /evidence="ECO:0000269|PubMed:11748244,
FT ECO:0000269|PubMed:19670200"
FT BINDING 172
FT /ligand="(2S,3S)-3-methyl-L-aspartate"
FT /ligand_id="ChEBI:CHEBI:58724"
FT /evidence="ECO:0000250"
FT BINDING 238
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000269|PubMed:11748244,
FT ECO:0000269|PubMed:22614383"
FT BINDING 273
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000269|PubMed:11748244,
FT ECO:0000269|PubMed:22614383"
FT BINDING 307
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000269|PubMed:11748244,
FT ECO:0000269|PubMed:22614383"
FT BINDING 329
FT /ligand="(2S,3S)-3-methyl-L-aspartate"
FT /ligand_id="ChEBI:CHEBI:58724"
FT BINDING 360..361
FT /ligand="(2S,3S)-3-methyl-L-aspartate"
FT /ligand_id="ChEBI:CHEBI:58724"
FT /evidence="ECO:0000250"
FT BINDING 361
FT /ligand="(2S,3S)-3-methyl-L-aspartate"
FT /ligand_id="ChEBI:CHEBI:58724"
FT SITE 194
FT /note="Transition state stabilizer"
FT MUTAGEN 73
FT /note="Q->A: It has very broad nucleophile scope and
FT excellent regio- and diastereoselectivity in the amination
FT reaction. This mutation strongly moves the specificity of
FT MAL away from ammonia and towards methylamine. It is highly
FT enantioselective."
FT /evidence="ECO:0000269|PubMed:22614383"
FT MUTAGEN 194
FT /note="H->A: Strong (160-fold) decrease of the catalytic
FT efficiency for deamination and slight (1.8-fold) decrease
FT of affinity binding for L-threo-beta-methylaspartate. 7-
FT fold decrease of the catalytic efficiency for amination and
FT 20-fold decrease of affinity binding for mesaconate. It
FT does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 194
FT /note="H->R: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 329
FT /note="Q->A: Very strong decrease of the catalytic
FT efficiency for deamination, whereas the affinity binding
FT for L-threo-beta-methylaspartate is not affected. Strong
FT (240-fold) decrease of the catalytic efficiency for
FT amination and slight (2.4-fold) decrease of affinity
FT binding for mesaconate. It does not show any major
FT conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 329
FT /note="Q->R: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 331
FT /note="K->A: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 331
FT /note="K->G: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 331
FT /note="K->H: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 331
FT /note="K->Q: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 331
FT /note="K->R: It abolishes deaminase and aminase activities
FT and does not show any major conformational changes."
FT /evidence="ECO:0000269|PubMed:19670200"
FT MUTAGEN 384
FT /note="L->A: It has very broad electrophile scope and
FT excellent regio- and enantioselectivity in the amination
FT reaction."
FT /evidence="ECO:0000269|PubMed:22614383"
FT STRAND 2..11
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 14..18
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 20..24
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 28..30
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 33..36
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 44..49
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 52..59
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 64..69
FT /evidence="ECO:0007829|PDB:1KCZ"
FT TURN 73..76
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 86..96
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 98..101
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 109..118
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 128..146
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 150..158
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 179..186
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 190..194
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 200..204
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 209..225
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 234..238
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 242..246
FT /evidence="ECO:0007829|PDB:1KCZ"
FT TURN 247..249
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 251..265
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 270..273
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 281..298
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 302..306
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 313..321
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 325..330
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 333..335
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 339..350
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 354..357
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 365..378
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 381..384
FT /evidence="ECO:0007829|PDB:1KCZ"
FT STRAND 387..391
FT /evidence="ECO:0007829|PDB:1KCZ"
FT HELIX 392..410
FT /evidence="ECO:0007829|PDB:1KCZ"
SQ SEQUENCE 413 AA; 45534 MW; 4451923DB035EF13 CRC64;
MKIVDVLCTP GLTGFYFDDQ RAIKKGAGHD GFTYTGSTVT EGFTQVRQKG ESISVLLVLE
DGQVAHGDCA AVQYSGAGGR DPLFLAKDFI PVIEKEIAPK LIGREITNFK PMAEEFDKMT
VNGNRLHTAI RYGITQAILD AVAKTRKVTM AEVIRDEYNP GAEINAVPVF AQSGDDRYDN
VDKMIIKEAD VLPHALINNV EEKLGLKGEK LLEYVKWLRD RIIKLRVRED YAPIFHIDVY
GTIGAAFDVD IKAMADYIQT LAEAAKPFHL RIEGPMDVED RQKQMEAMRD LRAELDGRGV
DAELVADEWC NTVEDVKFFT DNKAGHMVQI KTPDLGGVNN IADAIMYCKA NGMGAYCGGT
CNETNRSAEV TTNIGMACGA RQVLAKPGMG VDEGMMIVKN EMNRVLALVG RRK
