Half-of-the-Sites Reactivity of the Castor Δ9-18:0-Acyl Carrier Protein Desaturase

Abstract

Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor D9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the D9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity.

SlMYB75, an MYB-type transcription factor, promotes anthocyanin accumulation and enhances volatile aroma production in tomato fruits

“…While ethylene is known to regulate fruit ripening positively through inducing ripening-associated genes and autocatalytic ethylene production 38 , two seemingly conflicting features of SlMYB75 -OE tomatoes are their high level of ethylene production and delayed ripening (Fig. 2).…”

Amino Acid Change in an Orchid Desaturase Enables Mimicry of the Pollinator’s Sex Pheromone

“…Evolutionary modeling suggests that, once a functional SAD5-A allele is formed, pollinator-mediated selection would rapidly increase its allele frequency [33]. Third, SAD proteins act as dimers and recent biochemical work has shown that only one desaturase subunit is active at a time and that heterodimers with one LOF and one functional subunit have the same net activity as homodimers with two functional subunits, whereas only LOF/LOF homodimers (25%) are inactive [40]. Thus, ancestor proteins of SAD5, or their combinations in heterozygotes, may well have possessed desaturase activity sufficient for alkene production and associated pollinator attraction.…”

Quaternary structure of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) controls its activity

“…Half-of-sites reactivity is a general phenomenon in enzymology reported in numerous homodimeric proteins (37)(38)(39)(40)(41)(42)(43)(44). The homodimeric crystal structures of ACMSD invariably show one active site being well-ordered and another somewhat disordered, making ACMSD a candidate for half-of-sites reactivity (8,24,27,28).…”