<i>Myrothecium verrucaria</i> Bilirubin Oxidase and Its Mutants for Potential Copper Ligands

Shimizu, Atsushi; Kwon, Jung‐Hee; Sasaki, Takashi; Satoh, Takanori; Sakurai, Nobuo; Sakurai, Takeshi; Yamaguchi, Shotaro; Samejima, Tatsuya

doi:10.1021/bi9819531

Cited by 99 publications

(94 citation statements)

References 39 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In line with this, the recombinant Coprinus cinereus laccase has been reported to lack the type II Cu ion, and one of the His residues to it switched to coordinate one of type III Cus (13). As a type III Cu mutant, we prepared a double mutant, His456.458Val (2). However, trinuclear center was empty and only the type I Cu site was occupied by a Cu ion.…”

mentioning

confidence: 80%

“…These plasmids were purified from E. coli cells by the alkali-SDS method, and 5 mg of DNA was introduced into the A. oryzae niaD strain by protoplast transformation (15). Transformants were cultured with a minimum volume of medium containing nitrate as the sole nitrogen source for 5 days and further cultured for 5 days in the medium containing soybean oil, as reported (2,3). The level of the expression products of the Val mutant was moderate but those of the Asp and Lys mutants were high, as ascertained by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

“…In previous papers we reported the successful preparation of a variety of M. verrucaria BO mutants as to every Cu site and their characterization (2,3). The direct mutations at the type I Cu site (Met467Gly, Met467Gln, Met467His, Met467Arg, Cys457Ala, Cys457Val, His462Gly, and His462Val) indicated that Met467 is not necessarily indispensable for formation of the type I Cu center, as shown by mutation studies on blue copper proteins such as plastocyanin and azurin (11,12).…”

mentioning

confidence: 95%

“…BO is a multicopper oxidase containing one type I Cu, one type II Cu and a pair of type III Cus per protein molecule (2,3). Type I Cu mediates electron transfer from a substrate to other Cu sites.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

Shimizu¹

2003

Journal of Biochemistry

View full text Add to dashboard Cite

Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.

show abstract

mentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 95%

mentioning

confidence: 99%

See 2 more Smart Citations

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

Shimizu¹

2003

Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Two T3Cu signals with small A II values (15.0 x 10 -3 and 15.6 x 10 -3 cm -1 ) have been reported for a bilirubin oxidase mutant, His94Val, in which a His ligand to T2Cu was substituted by Val [15]. The Cu-OH-Cu structure will become more flexible because of the absence of the carboxyl group in the hydrogen bond network.…”

Section: Based On Spectral Property and Enzymatic Reactivity The Restmentioning

confidence: 97%

A novel resting form of the trinuclear copper center in the double mutant of a multicopper oxidase, CueO, Cys500Ser/Glu506Ala

Kajikawa

Sugiyama

Kataoka

et al. 2015

Journal of Inorganic Biochemistry

Self Cite

View full text Add to dashboard Cite

A multicopper oxidase, CueO was doubly mutated at its type I copper ligand, Cys500 and an acidic amino acid residue located in the proton transfer pathway, Glu506, to Ser and Ala, respectively.Cys500Ser/Glu506Ala was mainly in a novel resting form to afford the absorption band at ca. 400 nm and an EPR signal with a highly anisotropic character derived from type III copper. However, Cys500Ser/Glu506Ala gave the same reaction intermediate (peroxide intermediate) as that from Cys500Ser and Cys500Ser/Glu506Glu. with O 2 because one electron is deficient to form two water molecules [5]. On the other hand, the hydrogen bond network constructed with a conserved Glu residue (Glu506) and water molecules has been proved to function as a proton transport pathway from bulk water to TNC [6,8.9,[12][13][14] (Fig. 1). A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Mutations of this A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT4 the mutation, constructing a compensatory hydrogen bond network with only water molecules [9].In the present study we report unique spectral and magnetic properties of a double mutant of CueO, Cys500Ser/Glu506Ala at a ligand to T1Cu, Cys500 and at Glu506. The reaction of the double mutant with O 2 has also been performed to study properties of the intermediate I in comparison with that formed from Cys500Ser and Cys500Ser/Glu506Gln.Cys500Ser/Glu506Ala contained 3.2 Cu atoms in a protein molecule as determined from atomic absorption spectroscopy. Due to the absence of T1Cu the absorption bands at 600 nm disappeared, although the d-d band from TNC ( 745 ~400 M -1 cm -1 ) was observable ( Fig. 2A, full line).The band at 330 nm was considerably weakened ( ~2000 M -1 cm -1 relative to ~5200 M -1 cm -1 for the wild type enzyme (dotted line)), but a novel shoulder band was unequivocally observed at ca. 400 nm.This absorption band disappeared upon reduction, indicating that it did not come from an impurity.The corresponding circular dichrosim (CD) spectrum (Fig. 2B, full line) afforded a negatively signed band at 400 nm in addition to the weakened negatively signed band at 330 nm and the d-d bands at ca. 700 nm and 860 nm which were inverted in sign from those of Cys500Ser (dashed line). The EPR spectrum of Cys500Ser/Glu506Ala (Fig. 2C, full line) afforded a rhombic Cu(II) signal (g z = 2.40, g y = 2.11, g x = 2.04, A z = 7.6 x 10 -3 cm -1 , and A x = 5.3 x 10 -3 cm -1 ) differing from the resting CueO and Cys500Ser ( Fig. 2C dashed line). The EPR signal of T2Cu (g II = 2.25, A II = 19.6 x 10 -3 cm -1 ) was apparently minor. The total EPR-detectable Cu(II) in Cys500Ser/Glu500Ala was 2.2 per a protein molecule. Therefore, Cys500Ser/Glu506Ala is supposed to be in a unique resting state, both T3Cus to be magnetically isolated and T2Cu to be cuprous. Cys500Ser/Glu500Gln. Freezing of Cys500Ser/Glu506Ala soon after the reaction with O 2 gave the weak T2Cu EPR signal ( Supplementary Fig. 1, The total EPR-detectable Cu(II) was ~0.2 per a protein molecule), indicating that the main species ...

show abstract

Biocathodes for Dioxygen Reduction in Biofuel Cells

Bilewicz

Opałło

2010

Fuel Cell Science

View full text Add to dashboard Cite

Myrothecium verrucaria Bilirubin Oxidase and Its Mutants for Potential Copper Ligands

Cited by 99 publications

References 39 publications

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase

A novel resting form of the trinuclear copper center in the double mutant of a multicopper oxidase, CueO, Cys500Ser/Glu506Ala

Biocathodes for Dioxygen Reduction in Biofuel Cells

Contact Info

Product

Resources

About