Several weeks ago, a structural genomics consortium released a structure of a member of the ELOVL family of fatty acid elongases, specifically human ELOVL7. These multipass transmembrane proteins catalyze a reaction analogous to the ketoacyl synthase (KS) subunit of fatty acid synthase or the corresponding FabF enzyme in bacteria, which are soluble proteins--namely the decarboxylative Claisen condensation of a malonyl thioester with a straight chain acyl thioester to yield a beta-ketoacyl thioester. The thiol groups are provided by the ACP protein for fatty acid synthase/FabF and coenzyme A for the ELOVL proteins, but otherwise the reaction is identical.
The ACP-dependent, soluble elongases all use an active site cysteine side chain to accept the straight-chain acyl group from the "first" ACP molecule and transfer it to the acetyl enolate formed from the malonyl thioester:
However, the ELOVL proteins lack a conserved cysteine, which has made their mechanism unclear. What was known was that the most highly conserved sequence is a HXXHH motif in one of the transmembrane helices, which was tentatively proposed to bind a metal ion, and there are also several other highly conserved polar amino acids in the membrane-embedded regions (Hernandez-Buquer and Blacklock 2013).The use of a serine or threonine was proposed, or possibly a direct acyl transfer between two CoA groups simultaneously bound to the protein.
The new structure, which contains a bound product analog, clearly shows that there is only sufficient space for a single CoA group, and there is no metal ion present. Furthermore, the product analog forms two covalent bonds to the protein that suggest the catalytic mechanism. The precise structure of the analog is not provided in the PDB entry, but the coordinates are consistent with it having been a gamma-delta unsaturated, alpha-halo beta-ketoacyl CoA derivative. The first histidine of the HXXHH motif interacts with one of the amide groups of the CoA backbone and is unlikely to be a catalytic residue. The second histidine is hydrogen bonded to the third, and has undergone an apparent Michael addition to the delta position of the analog. Also, a fourth histidine remote in the primary sequence has displaced the presumed halide from the beta position to form a second covalent bond.
The location of that remote histidine (His 181), near the beta carbon and between the two carbonyl groups, suggests that it likely stabilizes the negative charge developing on the acetyl group during the decarboxylation step by hydrogen bond donation, and possibly also acts as the general acid that protonates the thiol leaving group in the acylation step.
The second histidine of the HXXHH motif (His 150), however, is deeper in the pocket (nearer the acyl tail) and together with the adjacent His 151 and an aspartate side chain on another helix, form a His-His-Asp triad reminiscent of the Ser-His-Asp triad of the serine proteases. Its position relative to the product analog, the membership in the triad, and the observed reactivity of this histidine strongly implicate it as the nucleophile that carries the transferring acyl group. This implies a mechanism similar to the following:
1. The long-chain acyl-CoA binds to the enzyme.
2. His-150 attacks the thioester carbonyl and retains the acyl group as an acyl-imidazole. Its nucleophilicity is enhanced by a proton relay, similar to the serine in serine proteases, and possibly by the ability of the developing positive charge to be shared across the two imidazole rings of the "HH" motif, which are relatively coplanar. An "oxyanion hole" formed by a lysine and an asparagine stabilizes the tetrahedral intermediate.
3. The released free CoA unbinds and malonyl-CoA binds.
4. Due to its carboxyl group being in a negatively charged pocket near Asp 130 and a nearby glutamate, and its acetyl fragment being near the His 181 NH proton, the malonyl group decarboxylates.
5. The resulting enolate attacks the His 150-bound acyl group, forming the product, in a step that may be separate or concerted with the decarboxylation.
The use of an acylhistidine intermediate is, to my knowledge, unprecedented in biology. Imidazoles as acyl transfer catalysts are, however, known from synthetic organic chemistry, for instance as part of the reagent carbonyldiimidazole. Phosphohistidine intermediates are relatively common in enzymes, appearing in several classes of phosphatase, but enzymes that transfer acyl groups have only been shown to use serine, cysteine, or occasionally threonine. However, approximately a year ago, Anthony Green and colleagues published the first successful de novo design of an artificial esterase using a histidine as a nucleophile(Burke et. al. 2019). It seems this may turn out to be one of the many cases in chemistry where nature "invented" something first. Comparison with the ELOVL7 structure offers an opportunity to compare how evolution and human design accomplish the same "trick".
In the case of Green's group's de novo esterase, a key requirement was methylation of the histidine to make the acylation reaction reversible. While methylated histidines are not unheard of in biology, in fact there's a "famous" example in the abundant protein actin, it appears that when used as a nucleophile, evolution has favored the use of an extended network of hydrogen bonded interactions instead.
The other interesting question is why this method of catalysis involving histidine is only used in this one example out of the many dozens of acyl-transferring enzymes that have been studied. Perhaps the use of an aromatic nuclophile, that can more easily be embedded in a desolvated transmembrane pocket than a small highly polar side chain, has favored its use here. All other intramembrane acyl transfer enzymes that I am aware of, such as protein palmitoyltransferases(PDB: 6BMS) and the MBOAT proteins(e.g. PDB: 6BUG), carry out direct attack of a soluble acceptor on a membrane-bound acyl-CoA molecule, without the need for an acyl-enzyme intermediate at all.
As this structure was determined by a structural genomics consortium as opposed to a dedicated lipid biosynthesis research group, I don't know how extensive the publication accompanying this structure will be once it is published. However, assuming that the authors agree on the implications of their structure, I am curious to see if they will present any biochemical data regarding the existence of the acylhistidine intermediate. All residues involved in the above mechanism are not only highly conserved, their mutation has (in most cases strongly) detrimental effects on catalysis in the ELOVL family member where it was tested (Hernandez-Buquer and Blacklock 2013), but this does not prove which residue carries the acyl group.
