Role of Cysteine Residues in the Structure, Stability, and Alkane Producing Activity of Cyanobacterial Aldehyde Deformylating Oxygenase

<div><p>Aldehyde deformylating oxygenase (AD) is a key enzyme for alkane biosynthesis in cyanobacteria, and it can be used as a catalyst for alkane production <i>in vitro</i> and <i>in vivo</i>. 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 <i>Nostoc punctiforme</i> 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.</p></div

Thermal denaturation curves of the wild type and mutant ADs.

<p>The denaturation curves were monitored by the CD ellipticity at 222 nm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122217#pone.0122217.s008" target="_blank">S8 Fig</a>). The values were then normalized to the baseline values of the native and unfolded states. A two-step denaturation was observed for the double mutants.</p

Crystal structure of <i>Prochlorococcus marinus</i> MIT9313 aldehyde deformylating oxygenase (<i>Pm</i>AD).

<p>Cys83, Cys119, and Ala129 in <i>Pm</i>AD, which corresponds to Cys71, Cys107, and Cys117 in AD from <i>Nostoc punctiforme</i> PCC 73102 (<i>Np</i>AD), respectively, are shown as red space-fill models (PDB ID: 2OC5). The two iron atoms and the substrate are shown as purple and yellow balls, respectively. The α-helices neighboring the helix involving Cys71 (cyan) are shown in blue and yellow-green. The figure was drawn using the PyMOL Molecular Graphics System, Schrödinger, LLC.</p

Activity, stability, and structural properties of the wild type and mutant aldehyde deformylating oxygenases (ADs).

<p>¹Hydrocarbon producing activity relative to that of the wild type.</p><p>²Expression level of AD protein, including the soluble and insoluble forms, in <i>Escherichia coli</i> is shown relative to that of the wild type.</p><p>³Solubility of AD when only AD was overexpressed in <i>E</i>. <i>coli</i> for <i>in vitro</i> characterization of the structure and stability. +: > 60% soluble; +/–: 20–60% soluble;–: < 20% soluble.</p><p>⁴Fraction of dimers (%), as estimated by size exclusion chromatography.</p><p>⁵Melting temperature, as measured by thermal denaturation. Errors are ±1°C. The <i>T</i>_m for the second transition is also shown for the double mutants in parenthesis.</p><p>⁶The means and standard deviations of duplicate or quadruplicate measurements are shown.</p><p>⁷not determined.</p><p>Activity, stability, and structural properties of the wild type and mutant aldehyde deformylating oxygenases (ADs).</p

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

<p>Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.</p

Hydrocarbon producing activities of the wild type and mutant ADs.

<p>The activity value presented here is relative to that of the wild type. The data are means ± standard deviations of duplicate or quadruplicate experiments.</p