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

Abstract

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 Polygonum minus 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 trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km 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 P. minus 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 P. minus 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.

Fig 1. Aldehydes substrates used for the substrate specificity of farnesal dehydrogenase from P. minus leaves.

Fig 2. Elution pattern of activity and protein of P. minus farnesal dehydrogenase from size exclusion column chromatography (Toyopearl TSK Gel G3000SW) for first time (A) and second time (B).

(●) Enzyme activity and (○) absorbance at 280 nm.

Fig 3. GC—MS analysis shows the purified P. minus farnesal dehydrogenase catalyse the farnesal into farnesoic acid.

(A) Separation of authentic farnesoic acid. The enzyme assay reaction containing subsrate farnesal, coenzyme NAD⁺ and with (B) and without (C) purified farnesal dehydrogenase. The Peak 2 indicates the retention time of 33.7 min for authentic farnesoic acid (A) and enzymatically produced farnesoic acid (B). The Peak 1 indicates the retention time of 30.9 min for the substrate farnesal.

Fig 4. Calibration curve on TSK-gel G3000SW and molecular mass of the farnesal dehydrogenase from P. minus.

(A) (●) Farnesal dehydrogenase (73 μg), (■) standard protein marker vitamin B₁₂ (1.35 kDa), myoglobin (17 kDa), ovalbumin (44 kDa) and γ-globulin (158 kDa). The elution pattern of the protein size markers was linear on a semilog plot. Elution data are represented as log molecular weight to Kav. Kav was calculated as in the equation (Ve-Vo)/(Vt-V0), Ve, Elution volume; Vo, Void volume; Vt, total column volume. (B) Purified enzyme and standard proteins were subjected to electrophoresis in the presence of SDS with 12.5% gel. Lane 1, molecular weight marker. Lane 2, purified farnesal dehydrogenase (6 μg). The arrow indicates the protein band shown on SDS-PAGE.

Fig 5. Effect of temperature towards activity of farnesal dehydrogenase.

(×) Temperature optimum, (●) residual activity after heat treatment. The optimal temperature was determined by performing the standard enzyme assay as described in “Materials and Methods,” except that the reaction temperature was varied. The effect of temperature on residual activity of the enzyme was determined by incubating the purified enzymes at a temperature in the range of 20–70°C for 10 min at pH 7.5 (100 mM tricine-NaOH containing 2.5 mM 2-ME). Relative activity values for temperature are indicated as mean values (n = 3).

Fig 6. Effects of pH on farnesal dehydrogenase activity.

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 (×), tris-HCl buffers (Δ), and glycine-NaOH buffers (■). Relative activity values for pH are indicated as mean values (n = 3).