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Q05514 (MAAL_CLOTT) Reviewed, UniProtKB/Swiss-Prot

Last modified July 9, 2014. Version 74. Feed History...

Clusters with 100%, 90%, 50% identity | Documents (3) | Third-party data text xml rdf/xml gff fasta
to top of pageNames·Attributes·General annotation·Ontologies·Sequence annotation·Sequences·References·Cross-refs·Entry info·DocumentsCustomize order

Names and origin

Protein namesRecommended name:
Methylaspartate ammonia-lyase

Short name=MAL
EC=4.3.1.2
Alternative name(s):
3-methylaspartase ammonia-lyase
Beta-methylaspartase
OrganismClostridium tetanomorphum
Taxonomic identifier1553 [NCBI]
Taxonomic lineageBacteriaFirmicutesClostridiaClostridialesClostridiaceaeClostridium

Protein attributes

Sequence length413 AA.
Sequence statusComplete.
Protein existenceEvidence at protein level

General annotation (Comments)

Function

Involved in the methylaspartate cycle. Catalyzes the formation of the alpha,beta-unsaturated bond by the reversible anti elimination of ammonia from L-threo-beta-methylaspartate (L-threo-(2S,3S)-3-methylaspartate) to give mesaconate. It can also use L-erythro-beta-methylaspartate (L-erythro-(2S,3R)-3-methylaspartate), L-aspartate, fumarate and ethylfumarate as substrates. Ref.1 Ref.5 Ref.6 Ref.8

Catalytic activity

L-threo-3-methylaspartate = mesaconate + NH3. Ref.1 Ref.5 Ref.6 Ref.8

Cofactor

Magnesium. Ref.5 Ref.7 Ref.8

Enzyme regulation

Inhibited by calcium ions. Ref.5

Pathway

Amino-acid degradation; L-glutamate degradation via mesaconate pathway; acetate and pyruvate from L-glutamate: step 2/4.

Subunit structure

Homodimer. Ref.1 Ref.6 Ref.7 Ref.8

Sequence similarities

Belongs to the methylaspartate ammonia-lyase family.

Biophysicochemical properties

Kinetic parameters:

Kcat is 61 sec(-1) for amination of mesaconate (with 20 mM of MgCl2 at pH 9 and at 30 degrees Celsius). Kcat is 89 sec(-1) for deamination of L-threo-beta-methylaspartate (with 20 mM of MgCl2 at pH 9 and at 30 degrees Celsius).

KM=0.65 mM for L-threo-beta-methylaspartate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius) Ref.1 Ref.5 Ref.6

KM=0.67 mM for L-threo-beta-methylaspartate (with 50 mM of KCl at pH 9 and at 30 degrees Celsius)

KM=0.7 mM for mesaconate (with 20 mM of MgCl2 at pH 9 and at 30 degrees Celsius)

KM=1 mM for L-threo-beta-methylaspartate (with 20 mM of MgCl2 at pH 9 and at 30 degrees Celsius)

KM=2.3 mM for L-aspartate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius)

KM=2.8 mM for L-threo-beta-methylaspartate (with 0.3 mM of KCl at pH 9 and at 30 degrees Celsius)

Vmax=2089 µmol/min/mg enzyme with L-threo-beta-methylaspartate as substrate (with 50 mM of KCl at pH 9 and at 30 degrees Celsius)

Vmax=309 µmol/min/mg enzyme with L-threo-beta-methylaspartate as substrate (with 0.3 mM of KCl at pH 9 and at 30 degrees Celsius)

Vmax=266 µmol/min/mg enzyme with L-threo-beta-methylaspartate as substrate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius)

Vmax=2.5 µmol/min/mg enzyme with L-erythro-beta-methylaspartate as substrate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius)

Vmax=2.4 µmol/min/mg enzyme with L-aspartate as substrate (with 4 mM of KCl at pH 9.76 and at 25 degrees Celsius)

pH dependence:

Optimum pH is 9.7.

Temperature dependence:

Optimum temperature is 55 degrees Celsius. It retains only half of its original activity after a 30 minutes incubation period at 50 degrees Celsius.

Sequence annotation (Features)

Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifier

Molecule processing

Chain1 – 413413Methylaspartate ammonia-lyase
PRO_0000084547

Regions

Region360 – 3612L-threo-beta-methylaspartate binding By similarity

Sites

Active site3311Proton acceptor Ref.6 Ref.7
Metal binding2381Magnesium
Metal binding2731Magnesium
Metal binding3071Magnesium
Binding site1721L-threo-beta-methylaspartate By similarity
Binding site3291L-threo-beta-methylaspartate
Binding site3611L-threo-beta-methylaspartate
Site1941Transition state stabilizer

Experimental info

Mutagenesis731Q → A: It has very broad nucleophile scope and excellent regio- and diastereoselectivity in the amination reaction. This mutation strongly moves the specificity of MAL away from ammonia and towards methylamine. It is highly enantioselective. Ref.8
Mutagenesis1941H → A: Strong (160-fold) decrease of the catalytic efficiency for deamination and slight (1.8-fold) decrease of affinity binding for L-threo-beta-methylaspartate. 7-fold decrease of the catalytic efficiency for amination and 20-fold decrease of affinity binding for mesaconate. It does not show any major conformational changes. Ref.6
Mutagenesis1941H → R: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3291Q → A: Very strong decrease of the catalytic efficiency for deamination, whereas the affinity binding for L-threo-beta-methylaspartate is not affected. Strong (240-fold) decrease of the catalytic efficiency for amination and slight (2.4-fold) decrease of affinity binding for mesaconate. It does not show any major conformational changes. Ref.6
Mutagenesis3291Q → R: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3311K → A: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3311K → G: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3311K → H: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3311K → Q: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3311K → R: It abolishes deaminase and aminase activities and does not show any major conformational changes. Ref.6
Mutagenesis3841L → A: It has very broad electrophile scope and excellent regio- and enantioselectivity in the amination reaction. Ref.8

Secondary structure

................................................................... 413
Helix Strand Turn

Details...

Sequences

Sequence LengthMass (Da)Tools
Q05514 [UniParc].

Last modified February 1, 1995. Version 1.
Checksum: 4451923DB035EF13

FASTA41345,534
        10         20         30         40         50         60 
MKIVDVLCTP GLTGFYFDDQ RAIKKGAGHD GFTYTGSTVT EGFTQVRQKG ESISVLLVLE 

        70         80         90        100        110        120 
DGQVAHGDCA AVQYSGAGGR DPLFLAKDFI PVIEKEIAPK LIGREITNFK PMAEEFDKMT 

       130        140        150        160        170        180 
VNGNRLHTAI RYGITQAILD AVAKTRKVTM AEVIRDEYNP GAEINAVPVF AQSGDDRYDN 

       190        200        210        220        230        240 
VDKMIIKEAD VLPHALINNV EEKLGLKGEK LLEYVKWLRD RIIKLRVRED YAPIFHIDVY 

       250        260        270        280        290        300 
GTIGAAFDVD IKAMADYIQT LAEAAKPFHL RIEGPMDVED RQKQMEAMRD LRAELDGRGV 

       310        320        330        340        350        360 
DAELVADEWC NTVEDVKFFT DNKAGHMVQI KTPDLGGVNN IADAIMYCKA NGMGAYCGGT 

       370        380        390        400        410 
CNETNRSAEV TTNIGMACGA RQVLAKPGMG VDEGMMIVKN EMNRVLALVG RRK 

« Hide

References

[1]"Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding beta-methylaspartase and characterization of the recombinant protein."
Goda S.K., Minton N.P., Botting N.P., Gani D.
Biochemistry 31:10747-10756(1992) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 1-26, FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT.
Strain: ATCC 15920 / DSM 528 / H1.
[2]"Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum."
Brecht M., Kellermann J., Plueckthun A.
FEBS Lett. 319:84-89(1993) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-24, PROTEIN SEQUENCE OF 1-24.
Strain: ATCC 15920 / DSM 528 / H1.
[3]"Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum. Organization of the mut genes."
Holloway D.E., Marsh E.N.G.
FEBS Lett. 317:44-48(1993) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-71.
Strain: NCIMB 11547.
[4]"Cloning and sequencing of glutamate mutase component S from Clostridium tetanomorphum. Homologies with other cobalamin-dependent enzymes."
Marsh E.N.G., Holloway D.E.
FEBS Lett. 310:167-170(1992) [PubMed] [Europe PMC] [Abstract]
Cited for: PROTEIN SEQUENCE OF 1-15.
Strain: NCIMB 11547.
[5]"The purification and properties of beta-methylaspartase."
Barker H.A., Smyth R.D., Wilson R.M., Weissbach H.
J. Biol. Chem. 234:320-328(1959) [PubMed] [Europe PMC] [Abstract]
Cited for: FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, ENZYME REGULATION, SUBSTRATE SPECIFICITY, COFACTOR.
Strain: ATCC 15920 / DSM 528 / H1.
[6]"Alteration of the diastereoselectivity of 3-methylaspartate ammonia lyase by using structure-based mutagenesis."
Raj H., Weiner B., Veetil V.P., Reis C.R., Quax W.J., Janssen D.B., Feringa B.L., Poelarends G.J.
ChemBioChem 10:2236-2245(2009) [PubMed] [Europe PMC] [Abstract]
Cited for: FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF HIS-194; GLN-329 AND LYS-331, SUBSTRATE SPECIFICITY, ACTIVE SITE, SUBUNIT.
Strain: ATCC 15920 / DSM 528 / H1.
[7]"The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step."
Asuncion M., Blankenfeldt W., Barlow J.N., Gani D., Naismith J.H.
J. Biol. Chem. 277:8306-8311(2002) [PubMed] [Europe PMC] [Abstract]
Cited for: X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) IN COMPLEX WITH MAGNESIUM, ACTIVE SITE, COFACTOR, SUBUNIT.
[8]"Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids."
Raj H., Szymanski W., de Villiers J., Rozeboom H.J., Veetil V.P., Reis C.R., de Villiers M., Dekker F.J., de Wildeman S., Quax W.J., Thunnissen A.M., Feringa B.L., Janssen D.B., Poelarends G.J.
Nat. Chem. 4:478-484(2012) [PubMed] [Europe PMC] [Abstract]
Cited for: X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) IN COMPLEX WITH SUBSTRATE ANALOGS AND MAGNESIUM, FUNCTION, CATALYTIC ACTIVITY, MUTAGENESIS OF GLN-73 AND LEU-384, COFACTOR, SUBUNIT.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ
S48141 Genomic DNA. Translation: AAB24070.1.
X70499 Genomic DNA. Translation: CAA49911.1.
X70695 Genomic DNA. Translation: CAA50027.1.
PIRB44285.

3D structure databases

PDBe
RCSB-PDB
PDBj
EntryMethodResolution (Å)ChainPositionsPDBsum
1KCZX-ray1.90A/B1-413[»]
1KD0X-ray1.90A/B1-413[»]
3ZVHX-ray1.99A/B1-413[»]
3ZVIX-ray1.90A/B1-413[»]
ProteinModelPortalQ05514.
SMRQ05514. Positions 1-413.
ModBaseSearch...
MobiDBSearch...

Protocols and materials databases

StructuralBiologyKnowledgebaseSearch...

Enzyme and pathway databases

BioCycMetaCyc:MONOMER-1103.
UniPathwayUPA00561; UER00618.

Family and domain databases

Gene3D3.20.20.120. 1 hit.
3.30.390.10. 1 hit.
InterProIPR029065. Enolase_C-like.
IPR029017. Enolase_N_like.
IPR006395. Me_Asp_am_lyase.
IPR022662. MeAsp_NH4-lyase_C.
IPR022665. MeAsp_NH4-lyase_N.
IPR001354. MR_MLE.
[Graphical view]
PANTHERPTHR13794. PTHR13794. 1 hit.
PfamPF07476. MAAL_C. 1 hit.
PF05034. MAAL_N. 1 hit.
[Graphical view]
PIRSFPIRSF017107. MAL. 1 hit.
SUPFAMSSF51604. SSF51604. 1 hit.
SSF54826. SSF54826. 1 hit.
TIGRFAMsTIGR01502. B_methylAsp_ase. 1 hit.
ProtoNetSearch...

Other

EvolutionaryTraceQ05514.

Entry information

Entry nameMAAL_CLOTT
AccessionPrimary (citable) accession number: Q05514
Entry history
Integrated into UniProtKB/Swiss-Prot: February 1, 1995
Last sequence update: February 1, 1995
Last modified: July 9, 2014
This is version 74 of the entry and version 1 of the sequence. [Complete history]
Entry statusReviewed (UniProtKB/Swiss-Prot)
Annotation programProkaryotic Protein Annotation Program

Relevant documents

SIMILARITY comments

Index of protein domains and families

PDB cross-references

Index of Protein Data Bank (PDB) cross-references

PATHWAY comments

Index of metabolic and biosynthesis pathways