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Literature summary 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

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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.8 gene FMO3, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain JM109 Homo sapiens
1.14.13.225 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)

EC Number Crystallization (Comment) Organism
1.14.13.225 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
1.14.13.225 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

EC Number Protein Variants Comment Organism
1.14.13.8 E158K naturally occuring polymorphic variant and site-directed mutagenesis, the melting temperature and activation energy of the mutant is nearly unaltered compared to the wild-type enzyme Homo sapiens
1.14.13.8 E308G naturally occuring polymorphic variant and site-directed mutagenesis, the mutant is unable to bind the NADP+ cofactor, it shows a significantly higher energy of unfolding (Ea) compared to wild-type Homo sapiens
1.14.13.8 additional information unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G are analyzed by differential scanning calorimetry (DSC) indicating that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the wild-type enzyme and its polymorphic variants is in a range from 46°C to 50°C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290-328 KJ/mol), except for the E308G variant that shows a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor stabilizes all the variants by shifting the main Tm by 4-5°C for all the proteins, exception made for E308G where no changes are observed Homo sapiens
1.14.13.8 V257M naturally occuring polymorphic variant and site-directed mutagenesis, the melting temperature and activation energy of the mutant is nearly unaltered compared to the wild-type enzyme Homo sapiens
1.14.13.225 E213G site-directed mutagenesis in the FMO domain Homo sapiens
1.14.13.225 E213G/R530G site-directed mutagenesis Homo sapiens
1.14.13.225 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
1.14.13.225 R530G site-directed mutagenesis in the CH domain Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.225 additional information
-
 additional information Michaelis-Menten kinetics Homo sapiens
1.14.13.225 0.2669
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCH Homo sapiens
1.14.13.225 0.5438
-
 [F-actin]-L-methionine recombinant MICAL3 containing the FMO and CH domains, pH 7.5, 25°C Homo sapiens
1.14.13.225 0.8172
-
 [F-actin]-L-methionine recombinant MICAL3 lacking the CH domain, pH 7.5, 25°C Homo sapiens
1.14.13.225 1.264
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMO Homo sapiens
1.14.13.225 1.438
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCHDELTA213,530 Homo sapiens

Localization

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

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.13.225 79400
-
 recombinant His-tagged enzyme, gel filtration Homo sapiens

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.8 Homo sapiens P31513
-
-
1.14.13.225 Homo sapiens Q7RTP6
-
-
1.14.13.225 Homo sapiens Q7RTP6 isoform MICAL3
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.8 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain JM109 by nickel affinity chromatography and ultrafiltration Homo sapiens
1.14.13.225 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

EC Number Source Tissue Comment Organism Textmining
1.14.13.225 skeletal muscle
-
 Homo sapiens
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.8 N,N-dimethylaniline + NADPH + H+ + O2
-
 Homo sapiens N,N-dimethylaniline N-oxide + NADP+ + H2O
-
 ?
1.14.13.225 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 ?
-
-
1.14.13.225 [F-actin]-L-methionine + NADPH + O2 + H+
-
 Homo sapiens [F-actin]-L-methionine-(R)-S-oxide + NADP+ + H2O
-
 ?
1.14.13.225 [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

EC Number Subunits Comment Organism
1.14.13.8 More heat-induced changes in the secondary structure in the presence and absence of NADP+, overview Homo sapiens
1.14.13.225 homodimer 2 * 40000, about, SDS-PAGE Homo sapiens
1.14.13.225 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

EC Number Synonyms Comment Organism
1.14.13.8 flavin-containing monooxygenase
-
 Homo sapiens
1.14.13.8 flavin-containing monooxygenase 3
-
 Homo sapiens
1.14.13.8 hFMO3
-
 Homo sapiens
1.14.13.225 MICAL3
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.8 25
-
 assay at Homo sapiens
1.14.13.225 25
-
 assay at Homo sapiens

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.14.13.8 46 50 differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible in all cases. The melting temperature (Tm) for the wild-type enzyme and its polymorphic variants is in a range from 46°C to 50°C at pH 7.4. Calculation for the activation energy of unfolding is performed using a mathematical model, secondary structures of wild-type and mutant enzymes during heat inactivation, overview Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.225 0.0006
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCH Homo sapiens
1.14.13.225 0.0024
-
 [F-actin]-L-methionine recombinant MICAL3 lacking the CH domain, pH 7.5, 25°C Homo sapiens
1.14.13.225 0.0031
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMO Homo sapiens
1.14.13.225 0.0033
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCHDELTA213,530 Homo sapiens
1.14.13.225 0.0169
-
 [F-actin]-L-methionine recombinant MICAL3 containing the FMO and CH domains, pH 7.5, 25°C Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.8 7.4
-
 assay at Homo sapiens
1.14.13.225 8
-
 assay at Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.8 FAD
-
 Homo sapiens
1.14.13.8 NADPH hFMO3 is reduced by its physiological electron donor NADPH. During the enzyme catalytic cycle, NADPH is consumed, then the reduced enzyme binds molecular oxygen to form a C4ahydroperoxy intermediate of the flavin responsible for the oxygenation of the substrate with the concomitant release of a water molecule. During these steps NADP+ remains bound in the active site leaving only when the catalytic cycle is completed. Heat-induced changes in the secondary structure in the presence and absence of NADP+, overview Homo sapiens
1.14.13.225 FAD
-
 Homo sapiens
1.14.13.225 NADPH
-
 Homo sapiens

General Information

EC Number General Information Comment Organism
1.14.13.8 malfunction the hFMO3 gene contains many naturally occuring single SNPs and these mutations can severely affect the activity of the enzyme resulting in lower or abolished activity Homo sapiens
1.14.13.8 additional information heat-induced changes in the secondary structure in the presence and absence of NADP+, overview Homo sapiens
1.14.13.8 physiological function human flavin-containing monooxygenases (hFMOs) comprise a family of five isoenzymes and are the second most important phase 1 drug-metabolizing enzymes after cytochromes P450. Its isoform 3 (hFMO3) is predominantly expressed in the liver where substrates containing nitrogen-, sulphur- and phosphorous-containing soft nucleophiles are transformed into more polar and excretable metabolites. Wild-type hFMO3 contributes to the metabolism of several important drugs such as ranitidine, cimetidine, tamoxifen, clozapine, benzydamine Homo sapiens
1.14.13.225 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
1.14.13.225 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
1.14.13.225 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.225 0.00225
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCH Homo sapiens
1.14.13.225 0.0023
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMOCHDELTA213,530 Homo sapiens
1.14.13.225 0.00245
-
 [F-actin]-L-methionine pH 8.0, 25°C, recombinant hMICAL3FMO Homo sapiens
1.14.13.225 0.0029
-
 [F-actin]-L-methionine recombinant MICAL3 lacking the CH domain, pH 7.5, 25°C Homo sapiens
1.14.13.225 0.031
-
 [F-actin]-L-methionine recombinant MICAL3 containing the FMO and CH domains, pH 7.5, 25°C Homo sapiens