Literature summary for 1.14.13.25 extracted from

Jones, J.C.; Banerjee, R.; Semonis, M.M.; Shi, K.; Aihara, H.; Lipscomb, J.D.
X-ray crystal structures of methane monooxygenase hydroxylase complexes with variants of its regulatory component correlations with altered reaction cycle dynamics (2022), Biochemistry, 61, 21-33 .

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Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Methylosinus trichosporium

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystallization of wild-type and mutant enzyme complexes by sitting drop vapour diffusion method, for the sMMOH:DBL2 and sMMOH:H5A proteins, 0.0015 ml of protein solution containing 0.06 mM sMMOH and 0.12 mM of either DBL2 or H5A in 100 mM MOPS buffer, pH 7.0, with 0.0015 ml cyrstallization solution containing 20% PEG 3350 and 0.2 M Na2HPO4, pH 8.8, equilibration against 0.5 ml of crystallization solution, 2-3 days at room temperature. For the sMMOH:DBL1 and sMMOH:H33A proteins, 0.0015 ml of protein solution containing 0.06 mM sMMOH and 0.12 mM of either DBL1 or H33A in 100 mM MOPS buffer, pH 7.0, with 0.0015 ml cyrstallization solution containing 21% PEG 3350 and 0.2 M Na2HPO4, pH 6.6, equilibration against 0.5 ml of crystallization solution, 2-3 days at room temperature, X-ray diffraction structure determination and analysis at 1.82-2.40 A resolution, structure modeling	Methylosinus trichosporium

Crystallization (Comment)

Organism

crystallization of wild-type and mutant enzyme complexes by sitting drop vapour diffusion method, for the sMMOH:DBL2 and sMMOH:H5A proteins, 0.0015 ml of protein solution containing 0.06 mM sMMOH and 0.12 mM of either DBL2 or H5A in 100 mM MOPS buffer, pH 7.0, with 0.0015 ml cyrstallization solution containing 20% PEG 3350 and 0.2 M Na2HPO4, pH 8.8, equilibration against 0.5 ml of crystallization solution, 2-3 days at room temperature. For the sMMOH:DBL1 and sMMOH:H33A proteins, 0.0015 ml of protein solution containing 0.06 mM sMMOH and 0.12 mM of either DBL1 or H33A in 100 mM MOPS buffer, pH 7.0, with 0.0015 ml cyrstallization solution containing 21% PEG 3350 and 0.2 M Na2HPO4, pH 6.6, equilibration against 0.5 ml of crystallization solution, 2-3 days at room temperature, X-ray diffraction structure determination and analysis at 1.82-2.40 A resolution, structure modeling

Methylosinus trichosporium

Protein Variants

Protein Variants	Comment	Organism
H33A	site-directed mutagenesis, an N-terminal region variant, structure analysis	Methylosinus trichosporium
H5A	site-directed mutagenesis, an N-terminal region variant, structure analysis	Methylosinus trichosporium
additional information	construction of N- and C-terminal truncation variants. The dramatic effects of specific mutations on specific steps of the reaction cycle include the (i) retarding O2-binding (MMOB DELTA2-29 and V41R), (ii) uncoupling the O2-activation reaction from hydrocarbon oxidation (MMOB DELTA126-138), (iii) relaxing the size-selective entry of hydrocarbon substrates (Quad, DBL2), (iv) disrupting the quantum-tunneling nature of the HAT reaction of Q with methane (Quad), and (v) significantly increasing or decreasing the rate constants of reaction cycle steps (H33A, DBL1, DELTA126-138, H5A, V41R, and V39R). Effect of the MMOB variants on small-molecule access tunnels, overview	Methylosinus trichosporium
N107G/S109A/S110A/T111A	site-directed mutagenesis, mutations in the core region, termed the Quad variant, structure analysis	Methylosinus trichosporium
N107G/S110A	site-directed mutagenesis, a binary derivative of the Quad variant, termed DBL1. The DBL1 mutation in MMOB leads to a loss of the S110 hydrogen bond with N214 of the sMMOH alpha-subunit, structure analysis	Methylosinus trichosporium
S109A/T111A	site-directed mutagenesis, a binary derivative of the Quad variant, termed DBL2, structure analysis	Methylosinus trichosporium

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
methane + NAD(P)H + H+ + O2	Methylosinus trichosporium	-	methanol + NAD(P)+ + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Methylosinus trichosporium	A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7	MMOH, MMOR, MMOB, and MMOD	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Methylosinus trichosporium

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
methane + NAD(P)H + H+ + O2	-	Methylosinus trichosporium	methanol + NAD(P)+ + H2O	-	?

Synonyms

Synonyms	Comment	Organism
sMMO	-	Methylosinus trichosporium
soluble methane monooxygenase	-	Methylosinus trichosporium

Cofactor

Cofactor	Comment	Organism	Structure
FAD	-	Methylosinus trichosporium
NAD(P)H	-	Methylosinus trichosporium

General Information

General Information	Comment	Organism
malfunction	mutations in the core region of MMOB and in the N- and C-termini cause dramatic changes in the rate constants for steps in the sMMOH reaction cycle	Methylosinus trichosporium
additional information	significant conformational changes must be imparted within sMMOH by the binding of MMOB. Small-molecule tunnel analysis, overview	Methylosinus trichosporium
physiological function	full activity of soluble methane monooxygenase (sMMO) depends upon the formation of a 1:1 complex of the regulatory protein MMOB with each alpha subunit of the (alphabetagamma)2 hydroxylase, sMMOH	Methylosinus trichosporium