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O13437 (FDH_CANBO) Reviewed, UniProtKB/Swiss-Prot

Last modified October 16, 2013. Version 71. Feed History...

Clusters with 100%, 90%, 50% identity | Documents (2) | 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:
Formate dehydrogenase

EC=1.2.1.2
Alternative name(s):
NAD-dependent formate dehydrogenase
Gene names
Name:FDH1
Synonyms:FDH, FDH3
OrganismCandida boidinii (Yeast)
Taxonomic identifier5477 [NCBI]
Taxonomic lineageEukaryotaFungiDikaryaAscomycotaSaccharomycotinaSaccharomycetesSaccharomycetalesmitosporic SaccharomycetalesCandida

Protein attributes

Sequence length364 AA.
Sequence statusComplete.
Protein existenceEvidence at protein level

General annotation (Comments)

Function

NAD+-dependent enzyme which catalyzes the final step in the menthanol oxidation pathway. Involved in detoxification of formate. Ref.1 Ref.6

Catalytic activity

Formate + NAD+ = CO2 + NADH. Ref.1 Ref.2 Ref.3 Ref.6 Ref.8

Enzyme regulation

Cu2+, Hg and p-chloromercuribenzoate are strong inhibitors of enzyme activity and Ca2+, Mg2+, Zn2+, Mn2+, Cd2+ and Sn2+ have no effect on activity indicating a cysteine residue in the protein is essential for enzyme activity or to maintain the proper structure of the enzyme. Nitrite and nitrate inhibit some enzyme activity, however cyanide, azide, thiocyanate and cyanate are strong inhibitors of the enzymic reaction. The inhibition of cyanide is competitive with formate and reversible. Ref.6

Subunit structure

Homodimer. Ref.6 Ref.8

Subcellular location

Cytoplasm By similarity UniProtKB Q08911.

Induction

Expression is strongly induced by methanol, but is completely repressed in the presence of glucose. However, methanol induced expression is equally strong in cells grown on glucose when formate, methylamine or choline is added. No expression is detected in cells grown on glycerol. When formate, methylamine or choline is added to the culture medium of glycerol- or glucose-grown cells, they exhibit an induction of FDH1 expression. Ref.1 Ref.6

Disruption phenotype

Is able to grow on methanol in a batch culture experiment, but its growth is greatly inhibited and a toxic level of formate accumulates in the medium. Formate is not detected in the medium in a methanol-limited chemostat culture but deletion mutant shows only one-fourth of the growth yield of the wild-type. Ref.1

Biotechnological use

Ideal catalyst for synthesizing chiral compounds of high enantiomeric purity from prochiral precursors due to a favorable thermodynamic equilibrium, the oxidation of formate to carbon dioxide while also reducing NAD to NADH. However, the necessesity for the presence of large quantities of the enzyme and its rapid inactivation under biotransformation conditions results in higher costs for the biocatalyst industry. In order to make this enzymatic reduction viable and to perform it on a larger scale a more efficient and cost effective process has been established. Site-directed mutagenesis has been effective in stabilizing this commercially important enzyme for its application in the biotransformation of trimethyl pyruvate to L-tert leucine. Ref.2 Ref.7

Sequence similarities

Belongs to the D-isomer specific 2-hydroxyacid dehydrogenase family. FDH subfamily. UniProtKB P33160

Biophysicochemical properties

Kinetic parameters:

KM=13 mM for formate (at 30 degrees Celsius and at pH 7.5) Ref.2 Ref.3 Ref.5 Ref.6 Ref.8

KM=0.09 mM for NAD (at 30 degrees Celsius and at pH 7.5) Ref.6

KM=5.6 mM for formate (at 30 degrees Celsius and at pH 7.5)

KM=0.045 mM for NAD (at 30 degrees Celsius and at pH 7.5) Ref.2

KM=2.42 mM for formate (at 25 degrees Celsius and at pH 7.5)

KM=0.04 mM for NAD (at 25 degrees Celsius and at pH 7.5) Ref.3

KM=2.4 mM for formate (at 25 degrees Celsius and at pH 7.6)

KM=0.04 mM for NAD (at 25 degrees Celsius and at pH 7.6) Ref.5

KM=20.0 mM for formate (at 20 degrees Celsius, at pH 7.5 and after 2 weeks of storage at 4 degrees Celsius in GF buffer)

KM=0.05 mM for NAD (at 20 degrees Celsius, at pH 7.5 and after 2 weeks of storage at 4 degrees Celsius in GF buffer) Ref.8

KM=35.0 mM for formate (at 20 degrees Celsius, at pH 7.5 and after 4 months of storage at 4 degrees Celsius in GF buffer)

KM=0.09 mM for NAD (at 20 degrees Celsius, at pH 7.5 and after 4 months of storage at 4 degrees Celsius in GF buffer) Ref.8

Vmax=6 µM/min/mg enzyme Ref.2

pH dependence:

Optimum pH is 7.5-8.5. Ref.6

Temperature dependence:

Broad temperature optima between 45 and 55 degrees Celsius. Reaction rate increases steeply up to 55 degrees Celsius. 50% of activity lost after incubation for 20 minutes at 57 degrees Celsius. Thermal stability increases in the presence of glycerol. Ref.2 Ref.6

Sequence annotation (Features)

Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifier

Molecule processing

Chain1 – 364364Formate dehydrogenase
PRO_0000393949

Regions

Nucleotide binding159 – 1668ATP Potential
Nucleotide binding174 – 1752NAD By similarity UniProtKB P33160
Nucleotide binding256 – 2583NAD By similarity UniProtKB P33160
Nucleotide binding311 – 3144NAD By similarity UniProtKB P33160
Region1 – 119119Catalytic By similarity Ref.8 UniProtKB P33160
Region120 – 312193Coenzyme-binding By similarity Ref.8 UniProtKB P33160
Region313 – 36452Catalytic By similarity Ref.8 UniProtKB P33160

Sites

Active site2581 By similarity UniProtKB P33160
Active site3111Proton donor By similarity UniProtKB P33160
Binding site2821NAD By similarity UniProtKB P33160

Amino acid modifications

Glycosylation1041N-linked (GlcNAc...) Potential

Natural variations

Natural variant91D → G in strain: 2.2159.
Natural variant50 – 512ET → GN in strain: 2.2159 and NCYC 1513.
Natural variant531E → V in strain: 2.2159 and NCYC 1513.
Natural variant561K → Q in strain: 2.2159 and NCYC 1513.
Natural variant791L → I in strain: 2.2159 and NCYC 1513.
Natural variant841N → K in strain: 2.2159 and NCYC 1513.
Natural variant1081K → R in strain: 2.2159.
Natural variant1451I → N in strain: 2.2159.
Natural variant1841L → V in strain: 2.2159 and NCYC 1513.
Natural variant2021E → D in strain: 2.2159 and NCYC 1513.
Natural variant3081M → T in strain: 2.2159.
Natural variant3251E → Q in strain: 2.2159 and NCYC 1513.

Experimental info

Mutagenesis231C → S: Slight increase in substrate affinity for formate but no change in affinity for NAD, 9 degrees Celsius decrease in thermal stability compared to the wild-type, significantly higher stability compared to wild-type under biotransformation conditions, significantly more stable in the presence of CuCl(2); when associated with A-262. Large increase in substrate affinity for formate but no significant change in affinity for NAD, 13 degrees Celsius decrease in thermal stability compared to the wild-type, significantly more stable in the presence of CuCl(2); when associated with V-262. No significant change in affinity for formate or NAD, 5 degrees Celsius decrease in thermal stability compared to the wild-type, significantly higher stability compared to wild-type under biotransformation conditions, and significantly more stable in the presence of CuCl(2). Ref.2
Mutagenesis471K → E: Slight increase in substrate affinity for formate and also affinity for NAD increases by half after 2 weeks. Also after 4 months affinity for formate increases by more than half and affinity for NAD increases by more than half. Retains 84% of residual activity after incubation for 20 minutes at a thermal inactivation temperature of 55 degrees Celsius in samples stored for 2 weeks compared to wild-type which loses 50% of its activity at 55 degrees Celsius. Ref.8
Mutagenesis691F → A: 2-fold decrease in substrate affinity for formate, but no significant change in affinity for NAD. A significant reduction in catalytic activity compared to the wild-type. Ref.3
Mutagenesis1191N → A: 94-fold decrease in substrate affinity for formate and 2700-fold decrease in substrate affinity for NAD. A significant reduction in catalytic activity compared to the wild-type; when associated with A-311. Ref.3
Mutagenesis1191N → H: 80-fold decrease in substrate affinity for formate and a 1250-fold decrease in substrate affinity for NAD. A significant reduction in catalytic activity compared to the wild-type. Ref.3
Mutagenesis1751I → A: 2-fold decrease in substrate affinity for formate and a 12-fold decrease in substrate affinity for NAD. A significant reduction in catalytic activity compared to the wild-type. Ref.3
Mutagenesis1971Q → L: 4-fold decrease in substrate affinity for formate but no significant change in affinity for NAD compared to the wild-type. Ref.3
Mutagenesis2581R → A: No catalytic activity. Ref.3
Mutagenesis2621C → A: Slight increase in substrate affinity for formate but no change in affinity for NAD, 9 degrees Celsius decrease in thermal stability compared to the wild-type, greater stability at a higher pH compared to the wild-type; when associated with S-23. Ref.2
Mutagenesis2621C → V: Large increase in substrate affinity for formate but no significant change in affinity for NAD, 13 degrees Celsius decrease in thermal stability compared to the wild-type; when associated with S-23. Great increase in substrate affinity for formate and NAD and 8 degrees Celsius decrease in thermal stability compared to the wild-type. Ref.2
Mutagenesis2871Q → A: 2-fold decrease in substrate affinity for formate and 3-fold decrease in substrate affinity for NAD compared to the wild-type; when associated with A-311. Ref.3
Mutagenesis2871Q → E: 380-fold decrease in substrate affinity for formate and 3-fold decrease in substrate affinity for NAD compared to the wild-type; when associated with T-288. No significant decrease in substrate affinity for formate but a 4-fold decrease in substrate affinity for NAD and a significant reduction in catalytic activity compared to the wild-type, a more acidic pH is seen than in the wild-type, preventing formate binding by a single ionization of a group compared to that of the wild-type. Ref.3
Mutagenesis2881P → T: 380-fold decrease in substrate affinity for formate and 3-fold decrease in substrate affinity for NAD compared to the wild-type; when associated with E-287. Ref.3
Mutagenesis3111H → A: 2-fold decrease in substrate affinity for formate and 3-fold decrease in substrate affinity for NAD compared to the wild-type; when associated with A-287. 93-fold decrease in substrate affinity for formate and 2700-fold decrease in substrate affinity for NAD, and a significant reduction in catalytic activity compared to the wild-type; when associated with A-119. Ref.3
Mutagenesis3111H → Q: 10-fold decrease in substrate affinity for formate and significant reduction in the catalytic activity compared to the wild-type. Ref.3
Mutagenesis3281K → V: A 75% increase in substrate affinity for formate after 2 weeks and a 50% increase in affinity for NAD. However, after 4 months the affinity for formate increases 7-fold and affinity for NAD increases by 2 thirds. Retains 70% of residual activity after incubation for 20 minutes at a thermal inactivation temperature of 55 degrees Celsius in samples stored for 2 weeks compared to wild-type which loses 50% of its activity at 55 degrees Celsius. Ref.8
Mutagenesis3601K → A: Exhibits no change in substrate affinity for formate, but shows a 4-fold decrease in substrate affinity for NAD implying that L-360 side chain forms strong interactions with the cofactor. A higher reaction rate is observed at an acidic and basic pH values. Ref.5
Sequence conflict19 – 235KLYGC → EKLYG AA sequence Ref.3

Secondary structure

........................................................................ 364
Helix Strand Turn

Details...

Sequences

Sequence LengthMass (Da)Tools
O13437 [UniParc].

Last modified January 1, 1998. Version 1.
Checksum: 1B30982E0D5B77E8

FASTA36440,370
        10         20         30         40         50         60 
MKIVLVLYDA GKHAADEEKL YGCTENKLGI ANWLKDQGHE LITTSDKEGE TSELDKHIPD 

        70         80         90        100        110        120 
ADIIITTPFH PAYITKERLD KAKNLKLVVV AGVGSDHIDL DYINQTGKKI SVLEVTGSNV 

       130        140        150        160        170        180 
VSVAEHVVMT MLVLVRNFVP AHEQIINHDW EVAAIAKDAY DIEGKTIATI GAGRIGYRVL 

       190        200        210        220        230        240 
ERLLPFNPKE LLYYDYQALP KEAEEKVGAR RVENIEELVA QADIVTVNAP LHAGTKGLIN 

       250        260        270        280        290        300 
KELLSKFKKG AWLVNTARGA ICVAEDVAAA LESGQLRGYG GDVWFPQPAP KDHPWRDMRN 

       310        320        330        340        350        360 
KYGAGNAMTP HYSGTTLDAQ TRYAEGTKNI LESFFTGKFD YRPQDIILLN GEYVTKAYGK 


HDKK 

« Hide

References

[1]"Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline."
Sakai Y., Murdanoto A.P., Konishi T., Iwamatsu A., Kato N.
J. Bacteriol. 179:4480-4485(1997) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [MRNA], PROTEIN SEQUENCE OF 2-45; 57-76; 87-103; 190-201; 207-236; 242-246; 292-326 AND 329-354, FUNCTION, CATALYTIC ACTIVITY, DISRUPTION PHENOTYPE, INDUCTION.
Strain: S2.
[2]"Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues."
Slusarczyk H., Felber S., Kula M.R., Pohl M.
Eur. J. Biochem. 267:1280-1289(2000) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [MRNA], PROTEIN SEQUENCE OF 1-15, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, BIOTECHNOLOGY, MUTAGENESIS OF CYS-23 AND CYS-262.
Strain: ATCC 32195.
[3]"Active-site characterization of Candida boidinii formate dehydrogenase."
Labrou N.E., Rigden D.J.
Biochem. J. 354:455-463(2001) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [MRNA], PROTEIN SEQUENCE OF 1-30 AND 132-140, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF PHE-69; ASN-119; ILE-175; GLN-197; ARG-258; GLN-287; PRO-288 AND HIS-311.
Strain: NCYC 1513.
[4]Zhang G., Yang G., Cao Z., Liu M.
Submitted (JUL-2007) to the EMBL/GenBank/DDBJ databases
Cited for: NUCLEOTIDE SEQUENCE [MRNA].
Strain: 2.2159.
[5]"Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis."
Labrou N.E., Rigden D.J., Clonis Y.D.
Eur. J. Biochem. 267:6657-6664(2000) [PubMed] [Europe PMC] [Abstract]
Cited for: PROTEIN SEQUENCE OF 357-363, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF LYS-360.
[6]"Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii."
Schute H., Flossdorf J., Sahm H., Kula M.R.
Eur. J. Biochem. 62:151-160(1976) [PubMed] [Europe PMC] [Abstract]
Cited for: FUNCTION, CATALYTIC ACTIVITY, ENZYME REGULATION, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT.
[7]"Continuous computer controlled production of formate dehydrogenase (FDH) and isolation on a pilot scale."
Weuster-Botz D., Paschold H., Striegel B., Gieren H., Kula M.R., Wandrey C.
Chem. Eng. Technol. 17:131-137(1994)
Cited for: BIOTECHNOLOGY.
[8]"High-resolution structures of formate dehydrogenase from Candida boidinii."
Schirwitz K., Schmidt A., Lamzin V.S.
Protein Sci. 16:1146-1156(2007) [PubMed] [Europe PMC] [Abstract]
Cited for: X-RAY CRYSTALLOGRAPHY (1.7 ANGSTROMS) OF MUTANT GLU-47 AND (1.55 ANGSTROMS) OF MUTANT VAL-328, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, CATALYTIC AND COENZYME-BINDING REGIONS, MUTAGENESIS OF LYS-47 AND LYS-328.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ
AF004096 Genomic DNA. Translation: AAC49766.1.
AJ245934 Genomic DNA. Translation: CAB54834.1.
AJ011046 Genomic DNA. Translation: CAA09466.2.
DQ458777 Genomic DNA. Translation: ABE69165.2.

3D structure databases

PDBe
RCSB PDB
PDBj
EntryMethodResolution (Å)ChainPositionsPDBsum
2FSSX-ray1.70A/B/C/D2-364[»]
2J6IX-ray1.55A/B/C/D2-364[»]
ProteinModelPortalO13437.
SMRO13437. Positions 2-353.
ModBaseSearch...
MobiDBSearch...

Proteomic databases

PRIDEO13437.

Protocols and materials databases

StructuralBiologyKnowledgebaseSearch...

Enzyme and pathway databases

BioCycMetaCyc:MONOMER-17206.
SABIO-RKO13437.

Family and domain databases

Gene3D3.40.50.720. 2 hits.
InterProIPR006139. D-isomer_2_OHA_DH_cat_dom.
IPR006140. D-isomer_2_OHA_DH_NAD-bd.
IPR016040. NAD(P)-bd_dom.
[Graphical view]
PfamPF00389. 2-Hacid_dh. 1 hit.
PF02826. 2-Hacid_dh_C. 1 hit.
[Graphical view]
PROSITEPS00065. D_2_HYDROXYACID_DH_1. 1 hit.
PS00670. D_2_HYDROXYACID_DH_2. 1 hit.
PS00671. D_2_HYDROXYACID_DH_3. 1 hit.
[Graphical view]
ProtoNetSearch...

Other

EvolutionaryTraceO13437.

Entry information

Entry nameFDH_CANBO
AccessionPrimary (citable) accession number: O13437
Secondary accession number(s): O93968, Q1PAH3
Entry history
Integrated into UniProtKB/Swiss-Prot: May 18, 2010
Last sequence update: January 1, 1998
Last modified: October 16, 2013
This is version 71 of the entry and version 1 of the sequence. [Complete history]
Entry statusReviewed (UniProtKB/Swiss-Prot)
Annotation programFungal Protein Annotation Program

Relevant documents

SIMILARITY comments

Index of protein domains and families

PDB cross-references

Index of Protein Data Bank (PDB) cross-references