User:Meglight/Phytoene desaturase (lycopene-forming)

Phytoene desaturase (lycopene-forming) (PDS) (CrtI, four-step phytoene desaturase) (EC 1.3.99.31, 15-cis-phytoene:acceptor oxidoreductase (lycopene-forming)) are enzymes found in archaea, bacteria and fungi that are involved in carotenoid biosynthesis. They catalyze the conversion of colorless 15-cis-phytoene into a bright red lycopene in a biochemical pathway called the poly-trans pathway. The same process in plants and cyanobacteria utilizes four separate enzymes in a poly-cis pathway.

Reaction Pathway

The conversion of phytoene to lycopene in plants and cyanobacteria (left) compared to bacteria and fungi(right).

Cyanobacteria and plants require four steps in carotenoid synthesis of 15-cis-Phytoene to all-trans-lycopene. Phytoene is the product of the enzymatic reaction involving phytoene synthase: phytoene synthase catalyzes the condensation reaction of GGPP (gernylgernyl pyrophosphate) to form phytoene. The lycopene formation is then catalyzed by four enzymes - PDS, Z-ISO (zeta-carotene isomerase), ZDS (zeta carotene desaturase), and CRTISO (carotene cis-trans isomerase) - in a four-step process. Phytoene is converted into zeta carotene through PDS catalysis. Z-ISO works in a transitional stage between PDS and ZDS catalysis, ZDS then aiding the formation of tetra-cis lycopene. CRTISO acts on the tetra-cis lycopene to produce all-trans-lycopene, which is later converted into carotenoid product.

Bacterial phytoene desaturase crtI functions as the only catalyzing agent in the formation of lycopene product from phytoene. During the chemical reaction, four additional double bonds are introduced into phytoene at locations 7, 11, 7' and 11':

15-cis-phytoene + 4 acceptor  ⇌ all-trans-lycopene + 4 reduced acceptor (overall reaction)

(1a) 15-cis-phytoene + acceptor ⇌   all-trans-phytofluene + reduced acceptor

(1b) all-trans-phytofluene + acceptor ⇌ all-trans-zeta-carotene + reduced acceptor

(1c) all-trans-zeta-carotene + acceptor ⇌  all-trans-neurosporene + reduced acceptor:

(1d) all-trans-neurosporene + acceptor ⇌  all-trans-lycopene + reduced acceptor

In contrast to PDS in cyanobacteria and plants, categorized as a dehydrogenase enzyme, PDS crtI is considered an oxidoreductase enzyme, requiring oxygen as a final electron acceptor in the reaction. 8 electrons are lost in total in the crtI enzymatic pathway. In anerobic conditions, quinones may also work as acceptor molecules in this reaction, duroquinone found to be the most effective in comparison to the efficiency of oxygen.

FAD Cofactor

Bacterial phytoene desaturases are shown to require FAD as a cofactor for their function . FAD, flavin adenine dinucleotide, acts as the redox cofactor in the transfer of electrons to oxygen. With FAD, the crtI protein gains catalytic efficiency; the crtI structure interacts with charges on the membrane bilayer and binds to the FAD cofactor to initiate carotenoid synthesis.

Results from a 2012 study indicate that FAD is the only successful cofactor for lycopene formation in bacterial cells. The PDS crtI enzyme was utilized from the bacteria Pantoea ananatis and expressed in the E. coli system to test crtI functional parameters. It was found that in the presence of cofactors FMN, NAD+, and NADP+, FAD was the only cofactor accepted by the PDS crtI enzyme for catalysis. Further tests revealed the structural need for FAD in lycopene production. Cells in which PDS – liposomal binding had occurred in the absence of FAD were not able to form lycopene. PDS crtI requires FAD as a binding cofactor for structural activation, otherwise remaining inactive if the cofactor is not present.

Structure

PDS crtI is comprised of beta – alpha sheets and includes three separate domains for substrate, membrane, and FAD binding. Altogether, the enzyme consists of “19 β-strands, 12 alpha-helices, and three 3₁₀-helices”.

The substrate binding domain structure includes “a seven-stranded mixed topology sheet with two alpha-helices packed onto the top surface and two, two-stranded anti-parallel sheets and two 3₁₀ helices packed onto one edge of the bottom surface of the sheet”.The domain consists of the active site for phytoene – enzyme association. The active center includes amino acid sites R148, D149, and R152. These sites are charged and hydrophobic, allowing for interaction with the hydrocarbon surface of phytoene through hydrogen transfer.

The membrane binding site structural form is a six-helix bundle. Such structure is interdependent on FAD binding structure. As discussed earlier, crtI – liposomal membrane association will occur in the absence of FAD, but the enzyme is be deemed inactive without the cofactor. Therefore, the FAD domain and membrane site shift in relation to one another, increasing entropy, or disorder, in the enzymatic system when FAD is present. This may change the structure of both binding sites upon activation.

The FAD binding domain is described as exhibiting a Rossman fold composed of various sheets for FAD binding: five β sheets lying between both a “three-stranded anti-parallel sheet and a five-helix bundle”. Both bacterial PDS and cyanobacteria/plant PDS contain such folding structure despite their molecular differences. The FAD binding site also relies on hydrophobic interactions with the FAD cofactor for association.

Cellular Function

It is thought tht the process of carotenoid formation from phytoene using PDS crtI in bacteria and fungi is tied to bacterial respiration.

Applications[edit]

In 2000 it was discovered that the gene insertion of a bacterial phytoene desaturase into transgenic tomatoes increased the lycopene content without the need to alter several of the plants enzymes. This approach was later used in rice to increase its β-carotene content resulting in the Golden Rice project.