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Role of cysteine residues in the structure, stability, and alkane producing activity of cyanobacterial aldehyde deformylating oxygenase.

Hayashi Y, Yasugi F, Arai M - PLoS ONE (2015)

Bottom Line: The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD.On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD.Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan.

ABSTRACT
Aldehyde deformylating oxygenase (AD) is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production in vitro and in vivo. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from Nostoc punctiforme PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

No MeSH data available.


Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.
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pone.0122217.g004: Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.

Mentions: The far-ultra violet (UV) CD spectra of the wild-type and mutant ADs revealed two minima at approximately 208 and 222 nm, typical of the CD spectra of proteins comprised mostly of α-helices (Fig 4). These results are consistent with the predominantly α-helical structure of PmAD (Fig 1). The spectra obtained for the mutant ADs were similar to that of the wild type, except in the case of C71A and C71A/C107A, indicating that overall structure was not affected by the mutations. However, the CD ellipticities of the C71A and C71A/C107A mutants were slightly higher and lower than that of the wild type, respectively. This might indicate small perturbations in secondary structure by the mutations and/or reflect uncertainties in the determination of protein concentrations.


Role of cysteine residues in the structure, stability, and alkane producing activity of cyanobacterial aldehyde deformylating oxygenase.

Hayashi Y, Yasugi F, Arai M - PLoS ONE (2015)

Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383598&req=5

pone.0122217.g004: Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.
Mentions: The far-ultra violet (UV) CD spectra of the wild-type and mutant ADs revealed two minima at approximately 208 and 222 nm, typical of the CD spectra of proteins comprised mostly of α-helices (Fig 4). These results are consistent with the predominantly α-helical structure of PmAD (Fig 1). The spectra obtained for the mutant ADs were similar to that of the wild type, except in the case of C71A and C71A/C107A, indicating that overall structure was not affected by the mutations. However, the CD ellipticities of the C71A and C71A/C107A mutants were slightly higher and lower than that of the wild type, respectively. This might indicate small perturbations in secondary structure by the mutations and/or reflect uncertainties in the determination of protein concentrations.

Bottom Line: The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD.On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD.Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan.

ABSTRACT
Aldehyde deformylating oxygenase (AD) is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production in vitro and in vivo. However, three free Cys residues in AD may impair its catalytic activity by undesired disulfide bond formation and oxidation. To develop Cys-deficient mutants of AD, we examined the roles of the Cys residues in the structure, stability, and alkane producing activity of AD from Nostoc punctiforme PCC 73102 by systematic Cys-to-Ala/Ser mutagenesis. The C71A/S mutations reduced the hydrocarbon producing activity of AD and facilitated the formation of a dimer, indicating that the conserved Cys71, which is located in close proximity to the substrate-binding site, plays crucial roles in maintaining the activity, structure, and stability of AD. On the other hand, mutations at Cys107 and Cys117 did not affect the hydrocarbon producing activity of AD. Therefore, we propose that the C107A/C117A double mutant is preferable to wild type AD for alkane production and that the double mutant may be used as a pseudo-wild type protein for further improvement of the alkane producing activity of AD.

No MeSH data available.