Effects of pH on farnesal dehydrogenase activity.

<p>Enzyme activity was assayed under the standard assay conditions, except that the following buffers were used at a final concentration of 100 mM in the incubation mixture: sodium citrate buffers (●), potassium phosphate buffers (<b>×</b>), tris-HCl buffers (Δ), and glycine-NaOH buffers (■). Relative activity values for pH are indicated as mean values (n = 3).</p

Novel NAD⁺-Farnesal Dehydrogenase from <i>Polygonum minus</i> Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

<div><p>Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD⁺-farnesal dehydrogenase of <i>Polygonum minus</i> was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD⁺. Other terpene aldehydes such as <i>trans</i>- cinnamaldehyde, citral and <i>α</i>- methyl cinnamaldehyde were also oxidized but in lower activity. The <i>K</i>_m values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD⁺ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative <i>P</i>. <i>minus</i> farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the <i>P</i>. <i>minus</i> farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant.</p></div

Aldehydes substrates used for the substrate specificity of farnesal dehydrogenase from <i>P</i>. <i>minus</i> leaves.

<p>Aldehydes substrates used for the substrate specificity of farnesal dehydrogenase from <i>P</i>. <i>minus</i> leaves.</p

Purification summary of farnesal dehydrogenase from <i>P</i>. <i>minus</i> leaves.

<p>Purification summary of farnesal dehydrogenase from <i>P</i>. <i>minus</i> leaves.</p

Effect of metal ions and inhibitors on farnesal dehydrogenase activity with farnesal as a substrate and NAD⁺ as a coenzyme.

<p>Effect of metal ions and inhibitors on farnesal dehydrogenase activity with farnesal as a substrate and NAD⁺ as a coenzyme.</p

Studies of substrate specificity, coenzyme specificity and kinetic parameters of purified farnesal dehydrogenase from <i>P</i>. <i>minus</i>.

<p>Studies of substrate specificity, coenzyme specificity and kinetic parameters of purified farnesal dehydrogenase from <i>P</i>. <i>minus</i>.</p

Elution pattern of activity and protein of <i>P</i>. <i>minus</i> farnesal dehydrogenase from size exclusion column chromatography (Toyopearl TSK Gel G3000SW) for first time (A) and second time (B).

<p>(●) Enzyme activity and (<b>○</b>) absorbance at 280 nm.</p

Identification of tryptic peptides from <i>P</i>. <i>minus</i> farnesal dehydrogenase.

<p>Identification of tryptic peptides from <i>P</i>. <i>minus</i> farnesal dehydrogenase.</p

Native-PAGE of the purified farnesol dehydrogenase from <i>P</i>. <i>minus</i>.

<p>Purified enzyme (33 μg) was subjected to electrophoresis in the absence of SDS with 12.5% gel at pH 8.8. Protein gel were stained by silver stain (A) and activity stain (B).</p

Substrate specificity, coenzyme specificity, and kinetic parameters of <i>P</i>. <i>minus</i> farnesol dehydrogenase.

<p>n.d- not determined.</p><p>Substrate specificity, coenzyme specificity, and kinetic parameters of <i>P</i>. <i>minus</i> farnesol dehydrogenase.</p