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 13-SEP-2023, entry version 109. 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; Bacillota; 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}. CC --------------------------------------------------------------------------- CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms CC Distributed under the Creative Commons Attribution (CC BY 4.0) License CC --------------------------------------------------------------------------- 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:MONOMER-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; Enolase-like C-terminal domain; 1. DR Gene3D; 3.30.390.10; Enolase-like, N-terminal domain; 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 NCBIfam; TIGR01502; B_methylAsp_ase; 1. DR PANTHER; PTHR48073:SF2; O-SUCCINYLBENZOATE SYNTHASE; 1. DR PANTHER; PTHR48073; O-SUCCINYLBENZOATE SYNTHASE-RELATED; 1. 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 SFLD; SFLDG00151; methylaspartate_ammonia-lyase; 1. DR SUPFAM; SSF51604; Enolase C-terminal domain-like; 1. DR SUPFAM; SSF54826; Enolase N-terminal domain-like; 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. FT 7-fold decrease of the catalytic efficiency for amination FT and 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 //