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Protein

4-hydroxy-2-oxovalerate aldolase 4

Gene

bphI

Organism
Burkholderia xenovorans (strain LB400)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Catalyzes the retro-aldol cleavage of both 4-hydroxy-2-oxopentanoate (HOPA) and 4-hydroxy-2-oxohexanoate (HOHA) to pyruvate and acetaldehyde or propanaldehyde, respectively. The aldehydes produced by this reaction are directly channeled from BphI to the dehydrogenase BphJ, ensuring that these toxic aldehydes are sequestered from cellular components. Is involved in the meta-cleavage pathway for the degradation of polychlorinated biphenyls (PCBs). Appears to be stereospecific since it can cleave (4S)-4-hydroxy-2-oxopentanoate but not the (4R) isomer. Also exhibits a secondary oxaloacetate decarboxylase activity. Finally, is also able to catalyze the reverse reaction, albeit much less efficiently, i.e. the condensation of aldehyde acceptors of two to three carbons in length with pyruvate. This aldol addition reaction is stereospecific; the condensation of acetaldehyde and pyruvate with BphI produces only the (4S)-4-hydroxy-2-oxopentanoate isomer. Aldehyde channeling in the BphI-BphJ complex can occur in reverse, from the dehydrogenase to the aldolase active sites, and the BphJ reductive deacylation reaction increases 4-fold when BphI is catalyzing the aldol addition reaction. Therefore, the BphI-BphJ enzyme complex exhibits unique bidirectionality in substrate channeling and allosteric activation.2 Publications

Catalytic activityi

(S)-4-hydroxy-2-oxopentanoate = acetaldehyde + pyruvate.1 Publication
(S)-4-hydroxy-2-oxohexanoate = propanal + pyruvate.1 Publication

Cofactori

Mn2+1 Publication, Cd2+1 Publication, Co2+1 PublicationNote: Divalent metal cation. Has the highest activity with Mn2+ as cofactor. Can also use Cd2+ at low concentrations (0.01-0.1 mM) or Co2+, although with less efficiency. Mg2+ and Ni2+ are very poor metal cofactors.1 Publication

Enzyme regulationi

Competitively inhibited by oxalate. Also inhibited by high concentrations of Cd2+ (1 mM) in vitro. Appears to be allosterically activated by aldehyde turnover occurring in BphJ, partly via NADH.

Kineticsi

The catalytic efficiency is similar when using 4-hydroxy-2-oxopentanoate or 4-hydroxy-2-oxohexanoate as substrate, but is 10-fold lower with 4-hydroxy-2-oxoheptanoate. It is also 25-fold higher when NADH is present than the value obtained without nucleotides. Moreover, the catalytic efficiency is similar when using acetaldehyde or propanaldehyde as substrate in the aldol addition reaction.

  1. KM=89 µM for (4S)-4-hydroxy-2-oxopentanoate (in the presence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  2. KM=0.22 mM for racemic 4-hydroxy-2-oxopentanoate (in the presence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  3. KM=1.12 mM for racemic 4-hydroxy-2-oxopentanoate (in the absence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  4. KM=0.18 mM for racemic 4-hydroxy-2-oxohexanoate (in the presence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  5. KM=0.35 mM for racemic 4-hydroxy-2-oxoheptanoate (in the presence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  6. KM=64.28 mM for acetaldehyde (in the absence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  7. KM=135.9 mM for propanaldehyde (in the absence of NADH at pH 8 and 25 degrees Celsius)3 Publications
  8. KM=13.0 mM for pyruvate (at pH 8 and 25 degrees Celsius)3 Publications

    pH dependencei

    Activity increases from pH 6.5 to 9.1 Publication

    Pathwayi: polychlorinated biphenyl degradation

    This protein is involved in the pathway polychlorinated biphenyl degradation, which is part of Xenobiotic degradation.
    View all proteins of this organism that are known to be involved in the pathway polychlorinated biphenyl degradation and in Xenobiotic degradation.

    Sites

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Sitei16 – 161Transition state stabilizer
    Metal bindingi17 – 171ManganeseBy similarity
    Active sitei20 – 201Proton acceptorSequence analysis
    Sitei87 – 871Important for aldehyde specificity, and governs stereochemical control
    Sitei89 – 891Important for aldehyde specificity; governs substrate alkyl chain length
    Binding sitei170 – 1701SubstrateBy similarity
    Metal bindingi199 – 1991Manganese; via tele nitrogenBy similarity
    Binding sitei199 – 1991SubstrateBy similarity
    Metal bindingi201 – 2011Manganese; via tele nitrogenBy similarity
    Binding sitei290 – 2901SubstrateBy similarity
    Sitei290 – 2901Governs stereochemical control

    GO - Molecular functioni

    GO - Biological processi

    Complete GO annotation...

    Keywords - Molecular functioni

    Lyase

    Keywords - Biological processi

    Aromatic hydrocarbons catabolism

    Keywords - Ligandi

    Cadmium, Cobalt, Manganese, Metal-binding

    Enzyme and pathway databases

    BioCyciBXEN266265:GJII-8839-MONOMER.
    BRENDAi4.1.3.39. 9987.
    4.1.3.43. 7691.
    SABIO-RKP51015.
    UniPathwayiUPA01002.

    Names & Taxonomyi

    Protein namesi
    Recommended name:
    4-hydroxy-2-oxovalerate aldolase 4 (EC:4.1.3.39)
    Short name:
    HOA 4
    Alternative name(s):
    4-hydroxy-2-keto-pentanoic acid aldolase 4
    4-hydroxy-2-oxohexanoate aldolase (EC:4.1.3.43)
    4-hydroxy-2-oxopentanoate aldolase 4
    Gene namesi
    Name:bphI
    Ordered Locus Names:Bxeno_C1121
    ORF Names:Bxe_C1187
    OrganismiBurkholderia xenovorans (strain LB400)
    Taxonomic identifieri266265 [NCBI]
    Taxonomic lineageiBacteriaProteobacteriaBetaproteobacteriaBurkholderialesBurkholderiaceaeParaburkholderia
    Proteomesi
    • UP000001817 Componenti: Chromosome 3

    Pathology & Biotechi

    Mutagenesis

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Mutagenesisi16 – 161R → A: Loss of aldol cleavage activity. 1 Publication
    Mutagenesisi16 – 161R → K: 4000-fold decrease in the catalytic efficiency of the aldol cleavage reaction. 1 Publication
    Mutagenesisi20 – 201H → A or S: 100-fold decrease in the catalytic efficiency of the aldol cleavage reaction. Dramatic reduction in acetaldehyde and propanaldehyde channeling efficiency by more than 70%. 2 Publications
    Mutagenesisi87 – 871L → A: 32-fold reduction in the catalytic efficiency with acetaldehyde as substrate of the aldol addition reaction, but no change in the catalytic efficiency using propanaldehyde; thus, exhibits a 40-fold preference for propanaldehyde over acetaldehyde. 2 Publications
    Mutagenesisi87 – 871L → N or W: Loss of aldolase activity (with either enantiomer of HOPA), but retains some decarboxylase activity for the smaller oxaloacetate substrate. In the retro-aldol cleavage reaction, is inactive toward 4(S)-HOPA but is active toward 4(R)-HOPA, albeit with a great reduction in catalytic efficiency, and in the aldol addition reaction, produces also exclusively the 4(R)-enantiomer; when associated with F-290. 2 Publications
    Mutagenesisi89 – 891L → A: As the wild-type enzyme, exhibits similar catalytic efficiency with acetaldehyde or propanaldehyde as substrate in the aldol addition reaction but displays higher catalytic efficiency with longer aldehydes (50-fold increase using pentaldehyde). Shows a reduction in aldehyde channeling efficiency by 30%. 2 Publications
    Mutagenesisi290 – 2901Y → F: Loss of stereochemical control as the mutant is able to catalyze the aldol cleavage of substrates with both R and S configurations at C4 with similar kinetic parameters. 3.5-fold decrease in the catalytic efficiency of the aldol cleavage reaction. Reduction in aldehyde channeling efficiency by more than 30%. In the retro-aldol cleavage reaction, is inactive toward 4(S)-HOPA but is active toward 4(R)-HOPA, albeit with a great reduction in catalytic efficiency, and in the aldol addition reaction, produces also exclusively the 4(R)-enantiomer; when associated with N-87 or W-87. 3 Publications
    Mutagenesisi290 – 2901Y → S: Loss of stereochemical control as the mutant is able to catalyze the aldol cleavage of substrates with both R and S configurations at C4 with similar kinetic parameters. 3.5-fold decrease in the catalytic efficiency of the aldol cleavage reaction. 3 Publications
    Mutagenesisi322 – 3221G → A: Displays a reduction in aldehyde channeling efficiency of about 20%. 1 Publication
    Mutagenesisi322 – 3221G → F or L: Unable to channel either acetaldehyde or propanaldehyde. 1 Publication
    Mutagenesisi323 – 3231G → A: Able to channel butyraldehyde (with less efficiency than wild-type) but not its isomer isobutyraldehyde. 1 Publication
    Mutagenesisi323 – 3231G → F: Unable to channel either acetaldehyde or propanaldehyde. 1 Publication
    Mutagenesisi323 – 3231G → L: Able to channel acetaldehyde but not the larger propanaldehyde. 1 Publication

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Chaini1 – 3463464-hydroxy-2-oxovalerate aldolase 4PRO_0000064978Add
    BLAST

    Interactioni

    Subunit structurei

    Heterotetramer composed of two BphI (aldolase) and two BphJ (dehydrogenase).1 Publication

    Protein-protein interaction databases

    STRINGi266265.Bxe_C1187.

    Structurei

    3D structure databases

    ProteinModelPortaliP51015.
    SMRiP51015. Positions 5-339.
    ModBaseiSearch...
    MobiDBiSearch...

    Family & Domainsi

    Region

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Regioni16 – 172Substrate bindingBy similarity

    Sequence similaritiesi

    Phylogenomic databases

    eggNOGiENOG4105DF3. Bacteria.
    COG0119. LUCA.
    HOGENOMiHOG000048047.
    KOiK18365.
    OMAiIDLYKIM.
    OrthoDBiEOG6C2WC0.

    Family and domain databases

    Gene3Di3.20.20.70. 1 hit.
    HAMAPiMF_01656. HOA.
    InterProiIPR017629. 4OH_2_O-val_aldolase.
    IPR013785. Aldolase_TIM.
    IPR012425. DmpG_comm.
    IPR000891. PYR_CT.
    [Graphical view]
    PANTHERiPTHR10277:SF3. PTHR10277:SF3. 1 hit.
    PfamiPF07836. DmpG_comm. 1 hit.
    PF00682. HMGL-like. 1 hit.
    [Graphical view]
    ProDomiPD005364. DmpG_comm. 1 hit.
    [Graphical view] [Entries sharing at least one domain]
    TIGRFAMsiTIGR03217. 4OH_2_O_val_ald. 1 hit.
    PROSITEiPS50991. PYR_CT. 1 hit.
    [Graphical view]

    Sequencei

    Sequence statusi: Complete.

    P51015-1 [UniParc]FASTAAdd to basket

    « Hide

            10         20         30         40         50
    MKLEGKKVTV HDMTLRDGMH PKRHQMTLEQ MKSIACGLDA AGIPLIEVTH
    60 70 80 90 100
    GDGLGGSSVN YGFPAHSDEE YLGAVIPLMK QAKVSALLLP GIGTVEHLKM
    110 120 130 140 150
    AKDLGVNTIR VATHCTEADV SEQHITQSRK LGLDTVGFLM MAHMASPEKL
    160 170 180 190 200
    VSQALLMQGY GANCIYVTDS AGYMLPDDVK ARLSAVRAAL KPETELGFHG
    210 220 230 240 250
    HHNLAMGVAN SIAAIEAGAT RIDAAAAGLG AGAGNTPMEV FIAVCARMGI
    260 270 280 290 300
    ETGVDVFKIQ DVAEDLVVPI MDHVIRIDRD SLTLGYAGVY SSFLLFAKRA
    310 320 330 340
    SAKYGVPARD ILVELGRRGM VGGQEDMIED TAMTMARERG LTLTAA
    Length:346
    Mass (Da):36,798
    Last modified:September 19, 2006 - v2
    Checksum:iBD4A951D513C97CE
    GO

    Experimental Info

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Sequence conflicti263 – 2631A → S in CAA54036 (PubMed:8026764).Curated
    Sequence conflicti302 – 3021A → E in CAA54036 (PubMed:8026764).Curated

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    X76500 Genomic DNA. Translation: CAA54036.1.
    CP000272 Genomic DNA. Translation: ABE37049.1.
    RefSeqiWP_003450974.1. NZ_CP008761.1.

    Genome annotation databases

    EnsemblBacteriaiABE37049; ABE37049; Bxe_C1187.
    GeneIDi4010699.
    KEGGibxb:DR64_8618.
    bxe:Bxe_C1187.
    PATRICi19343359. VBIBurXen52548_8937.

    Cross-referencesi

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    X76500 Genomic DNA. Translation: CAA54036.1.
    CP000272 Genomic DNA. Translation: ABE37049.1.
    RefSeqiWP_003450974.1. NZ_CP008761.1.

    3D structure databases

    ProteinModelPortaliP51015.
    SMRiP51015. Positions 5-339.
    ModBaseiSearch...
    MobiDBiSearch...

    Protein-protein interaction databases

    STRINGi266265.Bxe_C1187.

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Genome annotation databases

    EnsemblBacteriaiABE37049; ABE37049; Bxe_C1187.
    GeneIDi4010699.
    KEGGibxb:DR64_8618.
    bxe:Bxe_C1187.
    PATRICi19343359. VBIBurXen52548_8937.

    Phylogenomic databases

    eggNOGiENOG4105DF3. Bacteria.
    COG0119. LUCA.
    HOGENOMiHOG000048047.
    KOiK18365.
    OMAiIDLYKIM.
    OrthoDBiEOG6C2WC0.

    Enzyme and pathway databases

    UniPathwayiUPA01002.
    BioCyciBXEN266265:GJII-8839-MONOMER.
    BRENDAi4.1.3.39. 9987.
    4.1.3.43. 7691.
    SABIO-RKP51015.

    Family and domain databases

    Gene3Di3.20.20.70. 1 hit.
    HAMAPiMF_01656. HOA.
    InterProiIPR017629. 4OH_2_O-val_aldolase.
    IPR013785. Aldolase_TIM.
    IPR012425. DmpG_comm.
    IPR000891. PYR_CT.
    [Graphical view]
    PANTHERiPTHR10277:SF3. PTHR10277:SF3. 1 hit.
    PfamiPF07836. DmpG_comm. 1 hit.
    PF00682. HMGL-like. 1 hit.
    [Graphical view]
    ProDomiPD005364. DmpG_comm. 1 hit.
    [Graphical view] [Entries sharing at least one domain]
    TIGRFAMsiTIGR03217. 4OH_2_O_val_ald. 1 hit.
    PROSITEiPS50991. PYR_CT. 1 hit.
    [Graphical view]
    ProtoNetiSearch...

    Publicationsi

    « Hide 'large scale' publications
    1. "The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes."
      Hofer B., Backhaus S., Timmis K.N.
      Gene 144:9-16(1994) [PubMed] [Europe PMC] [Abstract]
      Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA].
      Strain: LB400.
    2. Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
      Strain: LB400.
    3. "Characterization of an aldolase-dehydrogenase complex that exhibits substrate channeling in the polychlorinated biphenyls degradation pathway."
      Baker P., Pan D., Carere J., Rossi A., Wang W., Seah S.Y.K.
      Biochemistry 48:6551-6558(2009) [PubMed] [Europe PMC] [Abstract]
      Cited for: FUNCTION, CATALYTIC ACTIVITY, COFACTOR, SUBSTRATE SPECIFICITY, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, COMPLEX WITH BPHJ.
      Strain: LB400.
    4. "Comparison of two metal-dependent pyruvate aldolases related by convergent evolution: substrate specificity, kinetic mechanism, and substrate channeling."
      Wang W., Baker P., Seah S.Y.
      Biochemistry 49:3774-3782(2010) [PubMed] [Europe PMC] [Abstract]
      Cited for: FUNCTION, SUBSTRATE SPECIFICITY, STEREOSPECIFICITY, KINETIC PARAMETERS, KINETIC MECHANISM.
      Strain: LB400.
    5. "Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI."
      Baker P., Carere J., Seah S.Y.
      Biochemistry 50:3559-3569(2011) [PubMed] [Europe PMC] [Abstract]
      Cited for: KINETIC PARAMETERS, MUTAGENESIS OF ARG-16; HIS-20; LEU-87; LEU-89 AND TYR-290, ACTIVE SITES, CATALYTIC MECHANISM.
      Strain: LB400.
    6. "Investigating the molecular determinants for substrate channeling in BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls degradation pathway."
      Carere J., Baker P., Seah S.Y.
      Biochemistry 50:8407-8416(2011) [PubMed] [Europe PMC] [Abstract]
      Cited for: MUTAGENESIS OF HIS-20; LEU-89; TYR-290; GLY-322 AND GLY-323, ALDEHYDE CHANNELING MECHANISM.
      Strain: LB400.
    7. "Rational design of stereoselectivity in the class II pyruvate aldolase BphI."
      Baker P., Seah S.Y.
      J. Am. Chem. Soc. 134:507-513(2012) [PubMed] [Europe PMC] [Abstract]
      Cited for: MUTAGENESIS OF LEU-87 AND TYR-290.
      Strain: LB400.

    Entry informationi

    Entry nameiHOA4_BURXL
    AccessioniPrimary (citable) accession number: P51015
    Secondary accession number(s): Q13FU0
    Entry historyi
    Integrated into UniProtKB/Swiss-Prot: October 1, 1996
    Last sequence update: September 19, 2006
    Last modified: June 8, 2016
    This is version 87 of the entry and version 2 of the sequence. [Complete history]
    Entry statusiReviewed (UniProtKB/Swiss-Prot)
    Annotation programProkaryotic Protein Annotation Program

    Miscellaneousi

    Miscellaneous

    The aldol addition reaction proceeds via a compulsory order mechanism, with pyruvate binding first.

    Keywords - Technical termi

    Allosteric enzyme, Complete proteome, Reference proteome

    Documents

    1. PATHWAY comments
      Index of metabolic and biosynthesis pathways
    2. SIMILARITY comments
      Index of protein domains and families

    Similar proteinsi

    Links to similar proteins from the UniProt Reference Clusters (UniRef) at 100%, 90% and 50% sequence identity:
    100%UniRef100 combines identical sequences and sub-fragments with 11 or more residues from any organism into one UniRef entry.
    90%UniRef90 is built by clustering UniRef100 sequences that have at least 90% sequence identity to, and 80% overlap with, the longest sequence (a.k.a seed sequence).
    50%UniRef50 is built by clustering UniRef90 seed sequences that have at least 50% sequence identity to, and 80% overlap with, the longest sequence in the cluster.