• A couple of kinases use ATP to convert mevalonate to its pyrophosphate.

  Rat Mevalonate Kinase with ATP Bound (1kvk)

• Mevalonate kinase is feedback inhibited by farnesyl phosphate, which binds in the ATP binding site

• The pyrophosphate then is decarboxylated by mevalonate pyrophosphate decarboxylase to get to the 5-carbon basic unit of the terpenes.

Mevalonate Decarboxylase (Yeast)	Isopentenyl Pyrophosphate Isomerase (H. sapiens), Substrate Analog Bound

• Many decarboxylases require thiamine (B₁) pyrophosphate as a cofactor; this one apparently does not.

• The putative binding site is shown as sticks.

• The IPP has a manganese (violet) at the active site; Cys139 and Glu207 are essential [J. Mol. Biol., 2007, 366, 1437, 1447].

A straightforward organic chemistry "curly arrow" mechanism can be written for the decarboxylation. [Write it yourself; think about what kinds of groups on the enzyme might be involved.]

The isomerization of the double bond uses an enzyme, of course, but we could do it easily in the laboratory. [Again, can you write an organic chemistry mechanism?]

Most of the pathway to squalene was worked out using the same techniques we discussed earlier. However, molecular biology approaches also have been tried [Newman and Chappell, Crit. Rev. in Biochem. Mol. Biol., 1999, 34, 95]:

Genetic Analysis to Identify Biosynthetic Pathways

These techniques rest in large part on three ideas:

• Many bacterial natural products are made by "assembly lines" of enzymes

• The genes for these enzymes are usually found on continuous stretches of DNA; that is, in operons
  • Inheritance of only a fraction of a pathway would convey no survival advantage

• Expression of these regions in alternative hosts confers biosynthetic competence

Back to the chemistry.

The isopentenyl and dimethylallyl pyrophosphates are in equilibrium

• The next step couples the two by an S_N1 reaction

• The product of this reaction then is coupled by another S_N1 to a second isopentenyl pyrophosphate

The enzymes accomplishing the couplings are called prenyl transferases [Review: Liang, et al, Eur. J. Biochem., 2002, 269, 3339]. Most organisms have one enzyme for each step; however, the avian enzyme does both steps.

• The enzyme must have at the active site a group that can remove the allylic H from isopentenyl pyrophosphate.

• It also requires groups that can stabilize a carbocation

• The rest of the binding cavity should be lilpophilic, to interact with the hydrocarbon chain

Farnesyl Synthase (1ubx) with Farnesyl Diphosphate Bound	Farnesyl Synthase (1uby) with DimethylAllyl Phosphate Bound
Closeup of Farnesyl Synthase Binding Pocket

Two structure features are important:

• The binding cavity is lined with non-polar, lipophilic residues (blue)

• At the end where the carbocation is formed, aspartate residues (yellow) sit on each side of the cavity; negative charges stabilizing the positive one electrostatically

• Mg ions (green) coordinate to the phosphate and to the aspartates, positioning the substrate

This is a strategy we will meet again.

The coupling is stereospecific:

• The pro-R hydrogen is abstracted

• The allyl cation adds to the si face of the double bond

The chain length of the product is controlled by two phenylalanine residues that block the bottom of the binding pocket:

Phe 112 and 113 Block the Bottom of the Pocket (1fps)	Phe 112-113 Mutated to Ala (1ubw)

With this mutation an enzyme capable of making geranyl pyrophosphate is converted into one that can make farnesyl pyrophosphate.

Two related notes:

The double bonds in all the compounds on this pathway are E- (trans). Enzymes exist that couple isoprene units and form cis double bonds.

• These enzymes can couple as many as 10-12 isoprene units

• The products are found cyclized, attached to quinones, and ultimately even in the visual pigments

Prenyl transferases exist that attach farnesyl or geranylgeranyl (20 carbons) units to proteins, tagging them as part of cell signaling systems, some of which are involved in cancer and bone resorption diseases

• The prenyl groups are attached to a C-terminal cysteine; the residue four amino acids inboard of the cysteine determines which prenyl group is attached

A cis-Prenyl Synthase from E. coli (1v7u)	The Ras Farnesyl Transferase, Farnesyl Analog Bound (1tn8)