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Protein

3-amino-5-hydroxybenzoate synthase

Gene

rifK

Organism
Amycolatopsis mediterranei (strain S699) (Nocardia mediterranei)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Catalyzes the dehydration and aromatization of 5-amino-5-deoxy-3-dehydroshikimate (aminoDHS) to 3-amino-5-hydroxybenzoate (AHBA), a compound that then serves as the starter unit for the assembly of a polyketide during the biosynthesis of rifamycin B and other ansamycin antibiotics. Can not utilize 5-deoxy-5-amino-3-dehydroquinate (aminoDHQ), 5-deoxy-5-aminoshikimate (aminoSA), quinate, 3-dehydroquinate, or 3-dehydroshikimate (DHS) as substrate.1 Publication
In a complex with RifL, RifK may have a second function in the AHBA pathway, acting as a transaminase introducing the nitrogen into the first pathway intermediate, UDP-3-keto-D-glucose, to give UDP-kanosamine. Appears to use glutamine as the nitrogen donor; NH4+ or asparagine are 30% less effective as nitrogen donors and neither glutamate nor aspartate show activity.1 Publication

Catalytic activityi

5-amino-5-deoxy-3-dehydroshikimate = 3-amino-5-hydroxybenzoate + H2O.1 Publication
UDP-3-keto-alpha-D-glucose + L-glutamine = UDP-alpha-D-kanosamine + L-glutaramate.1 Publication

Cofactori

pyridoxal 5'-phosphate2 PublicationsNote: Binds 1 pyridoxal phosphate per subunit.2 Publications

Enzyme regulationi

AHBA synthase activity is activated by 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP), an intermediate in the shikimate pathway, and is irreversibly inhibited by gabaculine (5-amino-1,3-cyclohexadiene-1-carboxylate).1 Publication

Kineticsi

kcat is 6.82 sec(-1) for AHBA synthase activity.1 Publication

  1. KM=0.164 mM for 5-amino-5-deoxy-3-dehydroshikimate2 Publications
  2. KM=0.122 mM for 5-amino-5-deoxy-3-dehydroshikimate2 Publications

    pH dependencei

    Optimum pH is 7.5 for AHBA synthase activity. Retains its activity over a broad range of pH. Over 84% of the maximum activity of AHBA synthase is maintained over a pH range from 7.0 to 9.0.1 Publication

    Temperature dependencei

    Optimum temperature is 33 degrees Celsius for AHBA synthase activity. Retains its activity over a broad range of temperature. Over 84% of the maximum activity of AHBA synthase is maintained over a temperature range from 28 to 50 degrees Celsius.1 Publication

    Pathway:irifamycin B biosynthesis

    This protein is involved in the pathway rifamycin B biosynthesis, which is part of Antibiotic biosynthesis.1 Publication
    View all proteins of this organism that are known to be involved in the pathway rifamycin B biosynthesis and in Antibiotic biosynthesis.

    GO - Molecular functioni

    GO - Biological processi

    Complete GO annotation...

    Keywords - Molecular functioni

    Lyase, Transferase

    Keywords - Biological processi

    Antibiotic biosynthesis

    Keywords - Ligandi

    Pyridoxal phosphate

    Enzyme and pathway databases

    BioCyciMetaCyc:MONOMER-14078.
    BRENDAi4.2.1.144. 313.
    UniPathwayiUPA01029.

    Names & Taxonomyi

    Protein namesi
    Recommended name:
    3-amino-5-hydroxybenzoate synthase (EC:4.2.1.144)
    Short name:
    AHBA synthase
    Alternative name(s):
    Putative UDP-kanosamine synthase aminotransferase subunit (EC:2.6.1.-)
    Gene namesi
    Name:rifK
    Synonyms:rifD
    Ordered Locus Names:RAM_03205, AMES_0625
    OrganismiAmycolatopsis mediterranei (strain S699) (Nocardia mediterranei)
    Taxonomic identifieri713604 [NCBI]
    Taxonomic lineageiBacteriaActinobacteriaPseudonocardialesPseudonocardiaceaeAmycolatopsis
    ProteomesiUP000006138 Componenti: Chromosome

    Pathology & Biotechi

    Disruption phenotypei

    Inactivation of the gene encoding AHBA synthase results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA. Cells lacking this gene do not accumulate aminoDHS, aminoDHQ, or their spontaneous aromatization product, protocatechuate.2 Publications

    Mutagenesis

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Mutagenesisi88 – 881F → A: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi159 – 1591D → A, E or K: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi162 – 1621H → L: 2-fold decrease in AHBA synthase activity. 1 Publication
    Mutagenesisi185 – 1851Q → E, H or L: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi188 – 1881K → D: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi219 – 2191R → A: 10-fold decrease in AHBA synthase activity. 1 Publication
    Mutagenesisi226 – 2261Y → F: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi236 – 2361R → A: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi291 – 2911Y → A: Loss of AHBA synthase activity. 1 Publication
    Mutagenesisi291 – 2911Y → F: 7-fold decrease in AHBA synthase activity. 1 Publication

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Initiator methioninei1 – 11Removed1 Publication
    Chaini2 – 3883873-amino-5-hydroxybenzoate synthasePRO_0000422400Add
    BLAST

    Amino acid modifications

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Modified residuei188 – 1881N6-(pyridoxal phosphate)lysine

    Interactioni

    Subunit structurei

    Homodimer. Can interact with RifL.1 Publication

    Structurei

    Secondary structure

    1
    388
    Legend: HelixTurnBeta strand
    Show more details
    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Helixi17 – 2812Combined sources
    Turni34 – 363Combined sources
    Helixi39 – 5012Combined sources
    Beta strandi54 – 607Combined sources
    Helixi62 – 7211Combined sources
    Beta strandi80 – 878Combined sources
    Helixi90 – 978Combined sources
    Beta strandi101 – 1055Combined sources
    Turni109 – 1113Combined sources
    Helixi116 – 1227Combined sources
    Beta strandi127 – 1304Combined sources
    Helixi135 – 1373Combined sources
    Helixi142 – 15211Combined sources
    Helixi173 – 1753Combined sources
    Beta strandi176 – 1783Combined sources
    Beta strandi180 – 1834Combined sources
    Beta strandi188 – 1903Combined sources
    Beta strandi192 – 1943Combined sources
    Beta strandi196 – 2005Combined sources
    Helixi205 – 21410Combined sources
    Helixi239 – 24911Combined sources
    Helixi252 – 27120Combined sources
    Beta strandi291 – 2966Combined sources
    Helixi302 – 31413Combined sources
    Beta strandi319 – 3213Combined sources
    Helixi326 – 3283Combined sources
    Helixi330 – 3345Combined sources
    Helixi342 – 3476Combined sources
    Helixi350 – 3589Combined sources
    Beta strandi359 – 3635Combined sources
    Helixi364 – 3685Combined sources
    Helixi371 – 38717Combined sources

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1B9HX-ray2.00A1-388[»]
    1B9IX-ray2.00A1-388[»]
    ProteinModelPortaliO52552.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiO52552.

    Family & Domainsi

    Sequence similaritiesi

    Belongs to the degT/dnrJ/eryC1 family.Curated

    Phylogenomic databases

    KOiK16016.

    Family and domain databases

    Gene3Di3.40.640.10. 1 hit.
    3.90.1150.10. 1 hit.
    InterProiIPR000653. DegT/StrS_aminotransferase.
    IPR015424. PyrdxlP-dep_Trfase.
    IPR015421. PyrdxlP-dep_Trfase_major_sub1.
    IPR015422. PyrdxlP-dep_Trfase_major_sub2.
    [Graphical view]
    PfamiPF01041. DegT_DnrJ_EryC1. 1 hit.
    [Graphical view]
    PIRSFiPIRSF000390. PLP_StrS. 1 hit.
    SUPFAMiSSF53383. SSF53383. 1 hit.

    Sequencei

    Sequence statusi: Complete.

    Sequence processingi: The displayed sequence is further processed into a mature form.

    O52552-1 [UniParc]FASTAAdd to basket

    « Hide

            10         20         30         40         50
    MNARKAPEFP AWPQYDDAER NGLVRALEQG QWWRMGGDEV NSFEREFAAH
    60 70 80 90 100
    HGAAHALAVT NGTHALELAL QVMGVGPGTE VIVPAFTFIS SSQAAQRLGA
    110 120 130 140 150
    VTVPVDVDAA TYNLDPEAVA AAVTPRTKVI MPVHMAGLMA DMDALAKISA
    160 170 180 190 200
    DTGVPLLQDA AHAHGARWQG KRVGELDSIA TFSFQNGKLM TAGEGGAVVF
    210 220 230 240 250
    PDGETEKYET AFLRHSCGRP RDDRRYFHKI AGSNMRLNEF SASVLRAQLA
    260 270 280 290 300
    RLDEQIAVRD ERWTLLSRLL GAIDGVVPQG GDVRADRNSH YMAMFRIPGL
    310 320 330 340 350
    TEERRNALVD RLVEAGLPAF AAFRAIYRTD AFWELGAPDE SVDAIARRCP
    360 370 380
    NTDAISSDCV WLHHRVLLAG EPELHATAEI IADAVARA
    Length:388
    Mass (Da):42,211
    Last modified:November 13, 2013 - v2
    Checksum:i6537B1541C0410F0
    GO

    Experimental Info

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Sequence conflicti56 – 561A → G in AAA75105 (PubMed:9497318).Curated
    Sequence conflicti262 – 2621R → P in AAC01720 (PubMed:9512878).Curated
    Sequence conflicti262 – 2621R → P in AAA75105 (PubMed:9497318).Curated
    Sequence conflicti386 – 3861A → G in AAC01720 (PubMed:9512878).Curated
    Sequence conflicti386 – 3861A → G in AAA75105 (PubMed:9497318).Curated

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    AF040570 Genomic DNA. Translation: AAC01720.1.
    U33061 Genomic DNA. Translation: AAA75105.1.
    CP002896 Genomic DNA. Translation: AEK39133.1.
    CP003729 Genomic DNA. Translation: AFO74161.1.
    PIRiI39599.
    RefSeqiWP_013222557.1. NC_018266.1.

    Genome annotation databases

    EnsemblBacteriaiAEK39133; AEK39133; RAM_03205.
    AFO74161; AFO74161; AMES_0625.
    KEGGiamm:AMES_0625.
    amn:RAM_03205.

    Cross-referencesi

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    AF040570 Genomic DNA. Translation: AAC01720.1.
    U33061 Genomic DNA. Translation: AAA75105.1.
    CP002896 Genomic DNA. Translation: AEK39133.1.
    CP003729 Genomic DNA. Translation: AFO74161.1.
    PIRiI39599.
    RefSeqiWP_013222557.1. NC_018266.1.

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1B9HX-ray2.00A1-388[»]
    1B9IX-ray2.00A1-388[»]
    ProteinModelPortaliO52552.
    ModBaseiSearch...
    MobiDBiSearch...

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Genome annotation databases

    EnsemblBacteriaiAEK39133; AEK39133; RAM_03205.
    AFO74161; AFO74161; AMES_0625.
    KEGGiamm:AMES_0625.
    amn:RAM_03205.

    Phylogenomic databases

    KOiK16016.

    Enzyme and pathway databases

    UniPathwayiUPA01029.
    BioCyciMetaCyc:MONOMER-14078.
    BRENDAi4.2.1.144. 313.

    Miscellaneous databases

    EvolutionaryTraceiO52552.

    Family and domain databases

    Gene3Di3.40.640.10. 1 hit.
    3.90.1150.10. 1 hit.
    InterProiIPR000653. DegT/StrS_aminotransferase.
    IPR015424. PyrdxlP-dep_Trfase.
    IPR015421. PyrdxlP-dep_Trfase_major_sub1.
    IPR015422. PyrdxlP-dep_Trfase_major_sub2.
    [Graphical view]
    PfamiPF01041. DegT_DnrJ_EryC1. 1 hit.
    [Graphical view]
    PIRSFiPIRSF000390. PLP_StrS. 1 hit.
    SUPFAMiSSF53383. SSF53383. 1 hit.
    ProtoNetiSearch...

    Publicationsi

    « Hide 'large scale' publications
    1. "Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699."
      August P.R., Tang L., Yoon Y.J., Ning S., Muller R., Yu T.W., Taylor M., Hoffmann D., Kim C.G., Zhang X., Hutchinson C.R., Floss H.G.
      Chem. Biol. 5:69-79(1998) [PubMed] [Europe PMC] [Abstract]
      Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA].
      Strain: S699.
    2. "3-amino-5-hydroxybenzoic acid synthase, the terminal enzyme in the formation of the precursor of mC7N units in rifamycin and related antibiotics."
      Kim C.G., Yu T.W., Fryhle C.B., Handa S., Floss H.G.
      J. Biol. Chem. 273:6030-6040(1998) [PubMed] [Europe PMC] [Abstract]
      Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 2-11; 74-90 AND 236-261, FUNCTION AS AHBA SYNTHASE, CATALYTIC ACTIVITY, COFACTOR, SUBSTRATE SPECIFICITY, BIOPHYSICOCHEMICAL PROPERTIES, ENZYME REGULATION, IDENTIFICATION BY MASS SPECTROMETRY, SUBUNIT, DISRUPTION PHENOTYPE, REACTION MECHANISM.
      Strain: S699.
    3. "Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699."
      Verma M., Kaur J., Kumar M., Kumari K., Saxena A., Anand S., Nigam A., Ravi V., Raghuvanshi S., Khurana P., Tyagi A.K., Khurana J.P., Lal R.
      J. Bacteriol. 193:5562-5563(2011) [PubMed] [Europe PMC] [Abstract]
      Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
      Strain: S699.
    4. "Complete genome sequence of Amycolatopsis mediterranei S699 based on de novo assembly via a combinatorial sequencing strategy."
      Tang B., Zhao W., Zheng H., Zhuo Y., Zhang L., Zhao G.P.
      J. Bacteriol. 194:5699-5700(2012) [PubMed] [Europe PMC] [Abstract]
      Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
      Strain: S699.
    5. "Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, the starter unit of rifamycin biosynthesis in amycolatopsis Mediterranei S699."
      Yu T.W., Muller R., Muller M., Zhang X., Draeger G., Kim C.G., Leistner E., Floss H.G.
      J. Biol. Chem. 276:12546-12555(2001) [PubMed] [Europe PMC] [Abstract]
      Cited for: DISRUPTION PHENOTYPE.
      Strain: S699.
    6. "The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), the precursor of mC7N units in ansamycin and mitomycin antibiotics: a review."
      Floss H.G., Yu T.W., Arakawa K.
      J. Antibiot. 64:35-44(2011) [PubMed] [Europe PMC] [Abstract]
      Cited for: PUTATIVE FUNCTION AS UDP-KANOSAMINE SYNTHASE, INTERACTION WITH RIFL, KINETIC PARAMETERS, PATHWAY, MUTAGENESIS OF PHE-88; ADP-159; HIS-162; GLN-185; LYS-188; ARG-219; TYR-226; ARG-236 AND TYR-291.
      Strain: S699.
    7. "Crystal structure of 3-amino-5-hydroxybenzoic acid (AHBA) synthase."
      Eads J.C., Beeby M., Scapin G., Yu T.W., Floss H.G.
      Biochemistry 38:9840-9849(1999) [PubMed] [Europe PMC] [Abstract]
      Cited for: X-RAY CRYSTALLOGRAPHY (2.00 ANGSTROMS) IN COMPLEXES WITH PYRIDOXAL PHOSPHATE AND THE INHIBITOR GABACULINE, COFACTOR, PYRIDOXAL PHOSPHATE AT LYS-188.
      Strain: S699.

    Entry informationi

    Entry nameiRIFK_AMYMS
    AccessioniPrimary (citable) accession number: O52552
    Secondary accession number(s): G0FS65, Q44095
    Entry historyi
    Integrated into UniProtKB/Swiss-Prot: May 29, 2013
    Last sequence update: November 13, 2013
    Last modified: July 22, 2015
    This is version 76 of the entry and version 2 of the sequence. [Complete history]
    Entry statusiReviewed (UniProtKB/Swiss-Prot)
    Annotation programProkaryotic Protein Annotation Program

    Miscellaneousi

    Miscellaneous

    Mechanistic studies show that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-amino-5-deoxy-3-dehydroshikimate and catalyzes both an alpha,beta-dehydration and a stereospecific 1,4-enolization of the substrate.1 Publication

    Keywords - Technical termi

    3D-structure, Complete proteome, Direct protein sequencing, Reference proteome

    Documents

    1. PATHWAY comments
      Index of metabolic and biosynthesis pathways
    2. PDB cross-references
      Index of Protein Data Bank (PDB) cross-references
    3. SIMILARITY comments
      Index of protein domains and families

    External Data

    Dasty 3

    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 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.