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Literature summary for 1.14.13.225 extracted from

  • Kim, J.; Lee, H.; Roh, Y.J.; Kim, H.U.; Shin, D.; Kim, S.; Son, J.; Lee, A.; Kim, M.; Park, J.; Hwang, S.Y.; Kim, K.; Lee, Y.K.; Jung, H.S.; Hwang, K.Y.; Lee, B.C.
    Structural and kinetic insights into flavin-containing monooxygenase and calponin-homology domains in human MICAL3 (2020), IUCrJ, 7, 90-99 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
additional information the catalytic efficiency of MICAL3 increases on adding F-actin only when the CH domain is available Homo sapiens

Cloned(Commentary)

Cloned (Comment) Organism
gene MICAL3, hMICAL3FMOCH, hMICAL3FMO, and hMICAL3FMODCHELTA213,530 are cloned into the pET-28b plasmid, recombinant overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta 2 pLysS Homo sapiens

Crystallization (Commentary)

Crystallization (Comment) Organism
MICAL3 has an FAD/NADP-binding Rossmann-fold domain for monooxygenase activity. The flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains of both MICAL3 and MICAL1 are highly similar in structure, but a different relative position of the calponin-homology domain in the asymmetric unit Homo sapiens
purified recombinant His-tagged wild-type and mutant MICAL3 variants, sitting drop vapor diffusion method, method optimization, mixing of 500 nl of 25 mg/ml protein in 50 mM Tris, pH 8.5, 100 mM NaCl, 1 mM 1,4-dithiothreitol, 1% glycerol, with 500 nl of crystallization solution containing 0.1 M bicine-NaOH, pH 9.2, 7% v/v MPD, one day, X-ray diffraction structure determination and analysis at 1.9-2.3 A resolution Homo sapiens

Protein Variants

Protein Variants Comment Organism
E213G site-directed mutagenesis in the FMO domain Homo sapiens
E213G/R530G site-directed mutagenesis Homo sapiens
additional information construction of enzyme mutants hMCAL3FMOCH, hMICAL3FMO and hMICAL3FMOCHDELTA213,530. The truncated form containing the FMO and CH domains is much more soluble than the full-length form but still retains catalytic activity for F-actin disassembly Homo sapiens
R530G site-directed mutagenesis in the CH domain Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Homo sapiens
0.2669
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCH Homo sapiens
0.5438
-
[F-actin]-L-methionine recombinant MICAL3 containing the FMO and CH domains, pH 7.5, 25°C Homo sapiens
0.8172
-
[F-actin]-L-methionine recombinant MICAL3 lacking the CH domain, pH 7.5, 25°C Homo sapiens
1.264
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMO Homo sapiens
1.438
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCHDELTA213,530 Homo sapiens

Localization

Localization Comment Organism GeneOntology No. Textmining
nucleus
-
Homo sapiens 5634
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
79400
-
recombinant His-tagged enzyme, gel filtration Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
[F-actin]-L-methionine + NADPH + O2 + H+ Homo sapiens
-
[F-actin]-L-methionine-(R)-S-oxide + NADP+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens Q7RTP6
-
-
Homo sapiens Q7RTP6 isoform MICAL3
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta 2 pLysS by nickel affinity chromatography, ultrafiltration, and gel filtration Homo sapiens

Source Tissue

Source Tissue Comment Organism Textmining
skeletal muscle
-
Homo sapiens
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information MICAL has additionally NADPH oxidase activity that underlies F-actin disassembly simultaneously with the oxidation of NADPH. For MICAL3 containing the FMO and CH domains, the kcat/Km ratio for NADPH oxidase activity increases dramatically on adding F-actin, while the kcat/Km value for MICAL3 lacking the CH domain changes little Homo sapiens ?
-
-
[F-actin]-L-methionine + NADPH + O2 + H+
-
Homo sapiens [F-actin]-L-methionine-(R)-S-oxide + NADP+ + H2O
-
?
[F-actin]-L-methionine + NADPH + O2 + H+ usage of F-actin from rabbit skeletal muscle purified from G-actin by ultracentrifugation Homo sapiens [F-actin]-L-methionine-(R)-S-oxide + NADP+ + H2O
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 40000, about, SDS-PAGE Homo sapiens
More human MICAL3 contains the flavin-containing monooxygenase (FMO) and calponin-homology (CH) domains, and has an FAD/NADP-binding Rossmann-fold domain for monooxygenase activity like MICAL1, structure comparisons of isozymes MICAL3 and MICAL1. The FMO and CH domains of both isozymes MICAL3 and MICAL1 are highly similar in structure, but superimposition of the two structures shows a different relative position of the CH domain in the asymmetric unit. The catalytic efficiency of MICAL3 dramatically increases on adding F-actin only when the CH domain is available Homo sapiens

Synonyms

Synonyms Comment Organism
MICAL3
-
Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0006
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCH Homo sapiens
0.0024
-
[F-actin]-L-methionine recombinant MICAL3 lacking the CH domain, pH 7.5, 25°C Homo sapiens
0.0031
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMO Homo sapiens
0.0033
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCHDELTA213,530 Homo sapiens
0.0169
-
[F-actin]-L-methionine recombinant MICAL3 containing the FMO and CH domains, pH 7.5, 25°C Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
FAD
-
Homo sapiens
NADPH
-
Homo sapiens

General Information

General Information Comment Organism
malfunction the catalytic efficiency of MICAL3 increases on adding F-actin only when the CH domain is available. But this does not occur when two residues, Glu213 and Arg530, are mutated in the FMO and CH domains, respectively Homo sapiens
additional information the catalytic efficiency of MICAL3 increases on adding F-actin only when the CH domain is available. MICAL3 is structurally highly similar to isozyme MICAL1, which suggests that they may adopt the same catalytic mechanism, but the difference in the relative position of the CH domain produces a difference in F-actin substrate specificity. Interaction analysis of the binding site between the CH domain and the FMO domain in human MICAL3, modeling, overview. The FMO-CH interaction in hMICAL3 is required to increase the catalytic efficiency by conferring specific binding to F-actin. The FMO domain that exhibits monooxygenase activity is localized at the N-terminus of MICAL and is highly conserved among species. The CH domain that is usually found in actin binding proteins is adjacent to the FMO domain and is also highly conserved. CH domains are classified into three types: types 1, 2, and 3. Whereas type 3 CH domains are mainly found in regulatory proteins associated with muscle contraction and signaling proteins, type 1 and 2 CH domains are usually found in cytoskeletal proteins. MICALs have a typical type 2 CH domain Homo sapiens
physiological function MICAL isozymes are involved in actin cytoskeleton reorganization through methionine oxidation. The enzyme functions in F-actin disassembly Homo sapiens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.00225
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCH Homo sapiens
0.0023
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCHDELTA213,530 Homo sapiens
0.00245
-
[F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMO Homo sapiens
0.0029
-
[F-actin]-L-methionine recombinant MICAL3 lacking the CH domain, pH 7.5, 25°C Homo sapiens
0.031
-
[F-actin]-L-methionine recombinant MICAL3 containing the FMO and CH domains, pH 7.5, 25°C Homo sapiens