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Protein

2,5-diketo-D-gluconic acid reductase A

Gene

dkgA

Organism
Corynebacterium sp. (strain ATCC 31090)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Catalyzes the reduction of 2,5-diketo-D-gluconic acid (25DKG) to 2-keto-L-gulonic acid (2KLG). 5-keto-D-fructose and dihydroxyacetone can also serve as substrates. 25DKGR-A exhibits a greater selectivity for the substrate and higher thermal stability than 25DKGR-B.

Catalytic activityi

2-dehydro-L-gulonate + NADP+ = 2,5-didehydro-D-gluconate + NADPH.

Enzyme regulationi

Inhibited by Zn2+, Fe3+, Cu2+ and Ni2+.

Kineticsi

  1. KM=26 mM for 2,5-diketo-D-gluconate1 Publication
  2. KM=10 µM for NADPH1 Publication

    pH dependencei

    Optimum pH is 6.4. Active over a broad pH range.1 Publication

    Sites

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Active sitei50 – 501Proton donor1 Publication
    Binding sitei108 – 1081Substrate1 Publication

    Regions

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Nucleotide bindingi188 – 24255NADP1 PublicationAdd
    BLAST

    GO - Molecular functioni

    GO - Biological processi

    Complete GO annotation...

    Keywords - Molecular functioni

    Oxidoreductase

    Keywords - Biological processi

    Ascorbate biosynthesis

    Keywords - Ligandi

    NADP

    Enzyme and pathway databases

    BioCyciMetaCyc:MONOMER-17833.
    BRENDAi1.1.1.274. 1664.
    1.1.1.346. 1664.
    SABIO-RKP06632.

    Names & Taxonomyi

    Protein namesi
    Recommended name:
    2,5-diketo-D-gluconic acid reductase A (EC:1.1.1.346)
    Short name:
    2,5-DKG reductase A
    Short name:
    2,5-DKGR A
    Short name:
    25DKGR-A
    Alternative name(s):
    AKR5C
    Gene namesi
    Name:dkgA
    OrganismiCorynebacterium sp. (strain ATCC 31090)
    Taxonomic identifieri268952 [NCBI]
    Taxonomic lineageiBacteriaActinobacteriaCorynebacterialesCorynebacteriaceaeCorynebacterium

    Subcellular locationi

    GO - Cellular componenti

    Complete GO annotation...

    Keywords - Cellular componenti

    Cytoplasm

    Pathology & Biotechi

    Biotechnological usei

    Introduced by genetic manipulation and expressed in Erwinia herbicola by Anderson et al. The resultant organism is able to convert in a single fermentative step D-glucose into 2-keto-L-gulonic acid, a key precursor in the industrial production of L-ascorbic acid (vitamin C). However, the technology still needs some working on and is not used in commercial production at present.

    Mutagenesis

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Mutagenesisi22 – 221F → Y: Tighter binding of NADH.
    Mutagenesisi232 – 2321K → G: Tighter binding of NADH.
    Mutagenesisi238 – 2381R → H: Tighter binding of NADH.
    Mutagenesisi272 – 2721A → G: Tighter binding of NADH.

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Initiator methioninei1 – 11Removed2 Publications
    Chaini2 – 2782772,5-diketo-D-gluconic acid reductase APRO_0000124597Add
    BLAST

    Interactioni

    Subunit structurei

    Monomer.1 Publication

    Structurei

    Secondary structure

    1
    278
    Legend: HelixTurnBeta strand
    Show more details
    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Beta strandi5 – 73Combined sources
    Beta strandi13 – 175Combined sources
    Helixi26 – 283Combined sources
    Helixi29 – 3911Combined sources
    Beta strandi43 – 453Combined sources
    Helixi47 – 493Combined sources
    Helixi54 – 6310Combined sources
    Helixi67 – 693Combined sources
    Beta strandi71 – 766Combined sources
    Helixi78 – 803Combined sources
    Helixi86 – 9712Combined sources
    Beta strandi102 – 1076Combined sources
    Helixi112 – 1143Combined sources
    Helixi117 – 12913Combined sources
    Beta strandi132 – 1409Combined sources
    Helixi143 – 15311Combined sources
    Beta strandi158 – 1636Combined sources
    Helixi171 – 1799Combined sources
    Beta strandi183 – 1886Combined sources
    Helixi191 – 1933Combined sources
    Helixi201 – 21010Combined sources
    Helixi214 – 22411Combined sources
    Helixi236 – 2438Combined sources
    Helixi252 – 2598Combined sources
    Beta strandi264 – 2663Combined sources
    Turni274 – 2763Combined sources

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1A80X-ray2.10A2-278[»]
    1HW6X-ray1.90A1-278[»]
    1M9HX-ray2.00A1-278[»]
    ProteinModelPortaliP06632.
    SMRiP06632. Positions 2-278.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiP06632.

    Family & Domainsi

    Sequence similaritiesi

    Belongs to the aldo/keto reductase family.Curated

    Family and domain databases

    Gene3Di3.20.20.100. 1 hit.
    InterProiIPR001395. Aldo/ket_red.
    IPR018170. Aldo/ket_reductase_CS.
    IPR020471. Aldo/keto_reductase_subgr.
    IPR023210. NADP_OxRdtase_dom.
    [Graphical view]
    PANTHERiPTHR11732. PTHR11732. 1 hit.
    PfamiPF00248. Aldo_ket_red. 1 hit.
    [Graphical view]
    PIRSFiPIRSF000097. AKR. 1 hit.
    PRINTSiPR00069. ALDKETRDTASE.
    SUPFAMiSSF51430. SSF51430. 1 hit.
    PROSITEiPS00798. ALDOKETO_REDUCTASE_1. 1 hit.
    [Graphical view]

    Sequencei

    Sequence statusi: Complete.

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

    P06632-1 [UniParc]FASTAAdd to basket

    « Hide

            10         20         30         40         50
    MTVPSIVLND GNSIPQLGYG VFKVPPADTQ RAVEEALEVG YRHIDTAAIY
    60 70 80 90 100
    GNEEGVGAAI AASGIARDDL FITTKLWNDR HDGDEPAAAI AESLAKLALD
    110 120 130 140 150
    QVDLYLVHWP TPAADNYVHA WEKMIELRAA GLTRSIGVSN HLVPHLERIV
    160 170 180 190 200
    AATGVVPAVN QIELHPAYQQ REITDWAAAH DVKIESWGPL GQGKYDLFGA
    210 220 230 240 250
    EPVTAAAAAH GKTPAQAVLR WHLQKGFVVF PKSVRRERLE ENLDVFDFDL
    260 270
    TDTEIAAIDA MDPGDGSGRV SAHPDEVD
    Length:278
    Mass (Da):30,119
    Last modified:January 23, 2007 - v3
    Checksum:i403A2E9AA4624A37
    GO

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    M12799 Genomic DNA. Translation: AAA83534.1.
    PIRiI40838.

    Cross-referencesi

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    M12799 Genomic DNA. Translation: AAA83534.1.
    PIRiI40838.

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1A80X-ray2.10A2-278[»]
    1HW6X-ray1.90A1-278[»]
    1M9HX-ray2.00A1-278[»]
    ProteinModelPortaliP06632.
    SMRiP06632. Positions 2-278.
    ModBaseiSearch...
    MobiDBiSearch...

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Enzyme and pathway databases

    BioCyciMetaCyc:MONOMER-17833.
    BRENDAi1.1.1.274. 1664.
    1.1.1.346. 1664.
    SABIO-RKP06632.

    Miscellaneous databases

    EvolutionaryTraceiP06632.

    Family and domain databases

    Gene3Di3.20.20.100. 1 hit.
    InterProiIPR001395. Aldo/ket_red.
    IPR018170. Aldo/ket_reductase_CS.
    IPR020471. Aldo/keto_reductase_subgr.
    IPR023210. NADP_OxRdtase_dom.
    [Graphical view]
    PANTHERiPTHR11732. PTHR11732. 1 hit.
    PfamiPF00248. Aldo_ket_red. 1 hit.
    [Graphical view]
    PIRSFiPIRSF000097. AKR. 1 hit.
    PRINTSiPR00069. ALDKETRDTASE.
    SUPFAMiSSF51430. SSF51430. 1 hit.
    PROSITEiPS00798. ALDOKETO_REDUCTASE_1. 1 hit.
    [Graphical view]
    ProtoNetiSearch...

    Publicationsi

    1. "Production of 2-keto-L-gulonate, an intermediate in L-ascorbate synthesis, by a genetically modified Erwinia herbicola."
      Anderson S., Marks C.B., Lazarus R.A., Miller J.V., Stafford K., Seymour J., Light D., Rastetter W., Estell D.A.
      Science 230:144-149(1985)
      Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 2-41; 76-89; 97-123 AND 184-194, CHARACTERIZATION.
    2. "Purification and characterization of 2,5-diketo-D-gluconate reductase from Corynebacterium sp."
      Miller J.V., Estell D.A., Lazarus R.A.
      J. Biol. Chem. 262:9016-9020(1987) [PubMed] [Europe PMC] [Abstract]
      Cited for: PROTEIN SEQUENCE OF 2-41, CHARACTERIZATION, BIOPHYSICOCHEMICAL PROPERTIES.
    3. "Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases."
      Khurana S., Sanli G., Powers D.B., Anderson S., Blaber M.
      Proteins 39:68-75(2000) [PubMed] [Europe PMC] [Abstract]
      Cited for: 3D-STRUCTURE MODELING OF SUBSTRATE-BINDING SITE.
    4. "Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1-A resolution."
      Khurana S., Powers D.B., Anderson S., Blaber M.
      Proc. Natl. Acad. Sci. U.S.A. 95:6768-6773(1998) [PubMed] [Europe PMC] [Abstract]
      Cited for: X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS).
    5. "Structural assembly of the active site in an aldo-keto reductase by NADPH cofactor."
      Sanli G., Blaber M.
      J. Mol. Biol. 309:1209-1218(2001) [PubMed] [Europe PMC] [Abstract]
      Cited for: X-RAY CRYSTALLOGRAPHY (1.9 ANGSTROMS), ACTIVE SITE.
    6. "Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase."
      Sanli G., Banta S., Anderson S., Blaber M.
      Protein Sci. 13:504-512(2004) [PubMed] [Europe PMC] [Abstract]
      Cited for: X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS) OF MUTANT WITH TYR-22; GLY-232; HIS-238 AND GLY-272 IN COMPLEX WITH NADH.

    Entry informationi

    Entry nameiDKGA_CORSC
    AccessioniPrimary (citable) accession number: P06632
    Entry historyi
    Integrated into UniProtKB/Swiss-Prot: November 1, 1988
    Last sequence update: January 23, 2007
    Last modified: June 24, 2015
    This is version 89 of the entry and version 3 of the sequence. [Complete history]
    Entry statusiReviewed (UniProtKB/Swiss-Prot)
    Annotation programProkaryotic Protein Annotation Program

    Miscellaneousi

    Miscellaneous

    Modeling study indicates that the active site may not be optimized for 2,5-diketo-D-gluconic acid.

    Keywords - Technical termi

    3D-structure, Direct protein sequencing

    Documents

    1. PDB cross-references
      Index of Protein Data Bank (PDB) cross-references
    2. 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.