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Information on EC 4.4.1.17 - Holocytochrome-c synthase and Organism(s) Saccharomyces cerevisiae and UniProt Accession P06182
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4.4.1.17 Holocytochrome-c synthaseIUBMB Comments
In the reverse direction, the enzyme catalyses the attachment of heme to two cysteine residues in the protein, forming thioether links.
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The taxonomic range for the selected organisms is: Saccharomyces cerevisiae
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
system iii, heme lyase, cytochrome c heme lyase, cyc2p, holocytochrome c synthase, holocytochrome c-type synthase, ccsa1, holocytochrome c synthetase, cytochrome c synthase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CCHL
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Cytochrome c heme-lyase
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Cytochrome c synthase
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Holocytochrome c synthetase
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Holocytochrome c-type synthase
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Holocytochrome-C synthase
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Holocytochrome-C-type synthase
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Synthetase, holocytochrome c
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
covalent attachment of heme
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SYSTEMATIC NAME
IUBMB Comments
holocytochrome-c apocytochrome-c-lyase (heme-forming)
In the reverse direction, the enzyme catalyses the attachment of heme to two cysteine residues in the protein, forming thioether links.
CAS REGISTRY NUMBER
COMMENTARY
75139-03-6
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
apo-iso-1 cytochrome c + heme
holocytochrome c
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single residue variants of five conserved N-terminal residues G6A, K10A, G11A, F15A and R18A of Saccharomyces cerevisiae iso-1 cytochrome c. F15A replacement, corresponding to F10 in the horse cytochrome c, is not matured at all. G6A, K10A, G11A, and R18A variants are matured
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Apocytochrome c + heme
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enzyme for the covalent attachment of heme to apocytochrome c
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Apocytochrome c + heme
Holocytochrome c
activity of the recombinant enzyme with several mutant variants of holocytochrome c552 from Hydrogenobacter thermophilus coexpressed in Escherichia coli in cytoplasm and periplasm, respectively, and enzyme activity with several cytocohrome variants from Equus caballus in Escherichia coli cells, heme attachment motifs, overview
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Apocytochrome c + heme
Holocytochrome c
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chimeric substrate comprising a short Saccharomyces cerevisiae mitochondrial cytochrome c N-terminal region plus the C-terminal sequence, including the CXXCH heme-binding motif, of Paracoccus denitrificans cytochrome c that is not otherwise processed by HCCS. Saccharomyces cerevisiae HCCS is able to attach heme to the chimeric protein
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Apocytochrome c + heme
Holocytochrome c
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chorse cytochrome c, recombinant substrate with mutations D2A, E4A, K5A, G6A, K7A, K8A and F10A. For the D2A, E4A and K7A variants, heme attachment is not attenuated by the amino acid replacements. The G6A and F10A variants are not matured at detectable levels.K5A and K8A variants of horse cytochrome c are also matured at similar levels to the wild type protein
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Apocytochrome c + heme
Holocytochrome c
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Equus caballus heart cytochrome c or Strep-tagged Saccharomyces cerevisiae cytochrome c are coexpressed in Escherichia coli strain BL21(DE3) with Saccharomyces cerevisiae holocytochrome c synthase. No product synthesis from truncated cytochrome c1 mutants G29X, H45X and K60X, with the X indicating the position of the inserted stop codon
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apocytochrome c1 + heme
holocytochrome c1
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apocytochrome c1 mutant with a CAPCH heme-binding site instead of the wild-type CAACH is substrate of holocytochrome-c synthase and strictly dependent upon the presence of putative redox protein Cyc2p for assembly. The identity of the second intervening residue in the CXXCH motif is the key in determining the holocytochrome-c synthase-dependent versus holocytochrome-c1 synthase-dependent assembly of holocytochrome c1
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additional information
?
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the enzyme shares the ability of CCM, i.e. System I, the cytochrome c maturation system, found in many bacteria and commonly employed in the maturation of bacterial cytochromes c in Escherichia coli-based expression systems, to mature hemes c with extended heme attachment motifs, comparison of System I and cytochrome c heme lyase, i.e. System III, substrate specificities, overview
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?
additional information
?
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Saccharomyces cerevisiae has another, similar enzyme: cytochrome-c1-heme lyase
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?
additional information
?
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no activity with cytochrome c1
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?
additional information
?
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the CXXCH motif and the N-terminus of the apocytochrome polypeptide are important protein-protein recognition motifs in enzyme-substrate interaction, the N-terminal region of the mitochondrial cytochrome c sequence is critical for attachment of heme to apocytochrome c
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Apocytochrome c + heme
?
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enzyme for the covalent attachment of heme to apocytochrome c
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?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
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Km values for holocytochrome c using mitochondria and mitoplasts from several yeast strains
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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enzyme activity increases after solubilization from the membrane with Triton X-100
additional information
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method for determination of activity by HPLC
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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enzyme is transported into the intermembrane space via a direct sorting pathway, that can occur in absence of external energy sources
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integral membrane protein, facing cytoplasmic surface
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separation of mitochondrial inner and outer membrane on a sucrose gradient
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integral membrane protein of inner membrane (facing cytoplasmic surface)
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
at least five heme c maturation systems have been identified: Systems I, II, III, IV, and V. System I, the cytochrome c maturation (CCM) system, involves genes ccmA-H5 and is found in various bacteria and in plant mitochondria. System I matures cytochromes c in the periplasmic space of bacteria. System II, cytochrome c synthesis (CCS), consists of four modular components and occurs in various bacteria as well as the thylakoid membranes of plants and algae. System III, also called cytochrome c heme lyase (CCHL), is a single enzyme found in the mitochondria of fungi, vertebrates and invertebrates
additional information
additional information
construction endogenous holocytochrome c mutant variant with a CX3CH motif. The second X residue in the heme binding motif of cyt c1 (CXXCH) is important for its recognition as a substrate by the enzyme
additional information
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the CP motifs, commonly found in heme-binding proteins, are not required for enzyme activity of te Saccharomyyces cerevisiae enzyme, while mutations in the human enzyme on the C-terminal side of the CP motifs cause disease states of microphthalmia with linear skin defects due to abolished or drastically attenuated holocytochrome c production
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
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comparison of amino acid sequence of Saccharomyces cerevisiae and Neurospora crassa enzymes to that of Saccharomyces cerevisiae cytochrome-c1-heme lyase
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C15A
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site-directed mutagenesis, cytoplasm-targeted mutant, the mutant is matured and undergoes heme attachment like the wild-type enzyme
C18A
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site-directed mutagenesis, cytoplasm-targeted mutant, the mutant does not undergo heme attachment
C26A
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site-directed mutagenesis of CP-motif 1 of the enzyme, the mutant shows activity similar to the wild-type enzyme
C26A/C42A
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site-directed mutagenesis of both CP-motifs of the enzyme, the mutant shows activity similar to the wild-type enzyme
C26A/P27A
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site-directed mutagenesis of CP-motif 1 of the enzyme, the mutant shows activity similar to the wild-type enzyme
C42A
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site-directed mutagenesis of CP-motif 2 of the enzyme, the mutant shows activity similar to the wild-type enzyme
C42A/P43A
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site-directed mutagenesis of CP-motif 2 of the enzyme, the mutant shows activity similar to the wild-type enzyme
E133A
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site-directed mutagenesis, the mutant shows highly reduced activity compared with the wild-type enzyme
E133K
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site-directed mutagenesis, the mutant shows highly reduced activity compared with the wild-type enzyme
F15A
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site-directed mutagenesis, periplasm-targeted mutant, the mutant variant does not produce holocytochrome c
F15E
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site-directed mutagenesis, periplasm-targeted mutant, the mutant variant does not produce holocytochrome c
F15I
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site-directed mutagenesis, periplasm-targeted mutant, the mutant shows reduced holocytochrome c production compared to the wild-type enzyme
F15W
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site-directed mutagenesis, periplasm-targeted mutant, the mutant shows reduced holocytochrome c production compared to the wild-type enzyme
F15Y
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site-directed mutagenesis, periplasm-targeted mutant, the mutant shows unaltered holocytochrome c production like the wild-type enzyme
H19K
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site-directed mutagenesis, cytoplasm-targeted mutant, the mutant does not undergo heme attachment
H19M
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site-directed mutagenesis, cytoplasm-targeted mutant, the mutant does not undergo heme attachment
H19R
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site-directed mutagenesis, cytoplasm-targeted mutant, the mutant does not undergo heme attachment
additional information
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construction of T17, K16, G29, H45, and K60 deletion mutants and of a mutant with periplasmic targeting sequence
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of the enzyme in Escherichia coli cytosplasm, coexpression with several mutant variants of holocytochrome c552 from Hydrogenobacter thermophilus, the latter are expressed in the periplasm, or several cytochrome c variants from Equus caballus
coexpression of the wild-type enzyme with periplasmic targeting sequence and addition of the Pseudomonas aeruginosa cytochrome c551/552 pre-sequence, and mutant enzymes with Equus caballus heart cytochrome c or Strep-tagged Saccharomyces cerevisiaecytochrome c in Escherichia coli strain BL21(DE3)
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recombinant expression of His-tagged wild-type enzyme and mutants in Escherichia coli strain BL21(DE3) cytoplasm, coexpression with Equus caballus cytochrome c
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
molecular biology
system III, cytochrome c heme lyase, is an enzyme found in the mitochondria of many eukaryotes, which is used for heterologous expression of mitochondrial holocytochromes c
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Enosawa, S.; Ohashi, A.
Localization of enzyme for heme attachment to apocytochrome c in yeast mitochindria
Biochem. Biophys. Res. Commun.
141
1145-1150
1986
Saccharomyces cerevisiae
Dumont, M.; Ernst, J.F.; Hampsey, D.M.; Sherman, F.
Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Sacchomyces cerevisiae
EMBO J.
6
235-241
1987
Saccharomyces cerevisiae
Zollner, A.; Rdel, G.; Haid, A.
Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-c1-heme lyase
Eur. J. Biochem.
207
1093-1100
1992
Saccharomyces cerevisiae, Neurospora crassa
Steiner, H.; Zollner, A.; Haid, A.; Neupert, W.; Lill, R.
Biogenesis of mitochondrial heme lyases in yeast. Import and folding in the intermembrane space
J. Biol. Chem.
270
22842-22849
1995
Saccharomyces cerevisiae
Tong, J.; Margoliash, E.
Cytochrome c heme lyase activity of yeast mitochondria
J. Biol. Chem.
273
25695-25702
1998
Saccharomyces cerevisiae
Bernard, D.G.; Gabilly, S.T.; Dujardin, G.; Merchant, S.; Hamel, P.P.
Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases
J. Biol. Chem.
278
49732-49742
2003
Homo sapiens (P53701), Homo sapiens, Mus musculus (P53702), Saccharomyces cerevisiae
Bernard, D.G.; Quevillon-Cheruel, S.; Merchant, S.; Guiard, B.; Hamel, P.P.
Cyc2p, a membrane-bound flavoprotein involved in the maturation of mitochondrial c-type cytochromes
J. Biol. Chem.
280
39852-39859
2005
Saccharomyces cerevisiae
Kemmer, D.; McHardy, L.M.; Hoon, S.; Reberioux, D.; Giaever, G.; Nislow, C.; Roskelley, C.D.; Roberge, M.
Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis
BMC Microbiol.
9
9-9
2009
Saccharomyces cerevisiae
Stevens, J.M.; Zhang, Y.; Muthuvel, G.; Sam, K.A.; Allen, J.W.; Ferguson, S.J.
The mitochondrial cytochrome c N-terminal region is critical for maturation by holocytochrome c synthase
FEBS Lett.
585
1891-1896
2011
Saccharomyces cerevisiae
Corvest, V.; Murrey, D.A.; Bernard, D.G.; Knaff, D.B.; Guiard, B.; Hamel, P.P.
c-type cytochrome assembly in Saccharomyces cerevisiae: a key residue for apocytochrome c1/lyase interaction
Genetics
186
561-571
2010
Saccharomyces cerevisiae
Moore, R.L.; Stevens, J.M.; Ferguson, S.J.
Mitochondrial cytochrome c synthase: CP motifs are not necessary for heme attachment to apocytochrome c
FEBS Lett.
585
3415-3419
2011
Saccharomyces cerevisiae
Zhang, Y.; Stevens, J.M.; Ferguson, S.J.
Substrate recognition of holocytochrome c synthase: N-terminal region and CXXCH motif of mitochondrial cytochrome c
FEBS Lett.
588
3367-3374
2014
Saccharomyces cerevisiae
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