FEATURE ARTICLE
The Molecular Anatomy of an Ancient Adaptive Event
Protein engineering identifies the structural basis of a 3.5 billion-year-old adaptation
Antony Dean
The Where in Structural Biology
In order to catalyze reactions, enzymes must first bind the substrates and coenzymes in crevices on their surfaces, called active sites. The part of the active site that we are interested in is called the coenzyme-binding pocket, for it is here that ICDH and IPMDH bind and discriminate between NADP and NAD (Figure 10). The coenzyme-binding pockets of ICDH and IPMDH are constructed from three loops, with a helix in ICDH being substituted by a sharp turn in IPMDH. This overall similarity in construction is a reflection of common ancestry (the coenzyme-binding pockets of unrelated dehydrogenases are structurally distinct) and the need to bind coenzymes that are structurally very similar. The ability to discriminate between NADP and NAD depends on the properties of the amino acid residues lining the pockets.
The processes of binding and discrimination are often likened to a lock and key, with an enzyme as a lock and the coenzymes (or substrates) as the keys. Only the correct key will fit snugly into the lock and allow a turn of the catalytic mechanism. The wrong key will not fit into the lock, or it will fit so poorly that turning the mechanism is difficult or even impossible. In this lock-and-key analogy, the amino acid residues lining the coenzyme-binding pocket are the lock’s tumblers. Just as real tumblers directly interact with the notches on keys, so these amino acid residues directly interact with chemical groups on the coenzymes. The coenzymes in question differ at only one group: In NADP an additional phosphate is attached to the ribose sugar at the 2' position, adjacent to the adenine ring (Figure 4). How amino acid residues lining the coenzyme binding pocket interact with the 2'-hydroxyl of NAD and the 2'-phosphate of NADP determines which of the coenzymes is used.
Three kinds of interactions are particularly important in making this determination: hydrogen bonds, electrostatic interactions and steric-packing effects. Hydrogen bonds are the same bonds that hold molecules of water together. They are sensitive to both the angle and the distance of the interacting atoms, which accounts for the ease of boiling water, whereby hydrogen bonds are broken to liberate free water molecules as gas. Electrostatic interactions occur between charged atoms. Oppositely charged atoms attract one another. For example, a crystal of common salt is held together by positively charged sodium ions interacting with negatively charged chloride ions. In contrast, atoms bearing similar charges repel one another: Static electricity makes hair stand on end as each charged strand repels its neighbors. Electrostatic interactions diminish with increasing distance, but remain insensitive to changes in angle. Steric-packing effects describe the physical fit of one molecule against another, much as the parts of a machine must fit snugly against one another.
In E. coli ICDH, hydrogen bonds form between the 2'-phosphate of NADP and two residues of the amino acid tyrosine (found at positions 345 and 391), a lysine residue (position 334), and two arginine residues (positions 395 and 292', where the prime designates residues of the second protein subunit). The lysine and the two arginine residues are positively charged and so attract the negatively charged 2'-phosphate of NADP. It is the pattern of hydrogen bonds and the electrostatic attraction that stabilize the binding of NADP to ICDH.
These same five amino acids are replaced by other residues in the NAD-dependent dehydrogenases. Here, all favorable interactions with the 2'-phosphate of NADP are eliminated: The sharp turn of IPMDH has no site equivalent to 395 in the helix of ICDH, whereas serine (292'), isoleucine (345) and proline (391) are incapable of hydrogen bonding to the 2'-phosphate of NADP. New, smaller amino acid residues at sites 345 and 351 eliminate steric overlap with NAD, allowing it to tilt to the left, and the ribose pucker to flip. This brings the ribose hydroxyls sufficiently close to aspartate (344) that hydrogen bonds form, stabilizing the NAD bound in the pocket. Not only do these hydrogen bonds stabilize the binding of NAD to IPMDH, but the negatively charged aspartate actively repels the negatively charged 2'-phosphate of NADP.
The critical five amino acid residues that interact with the 2'-phosphate are conserved in all known sequences of bacterial NADP-dependent ICDHs. Furthermore, algorithms designed to infer ancestral sequences from a known phylogeny reveal that these same five amino acid residues were present at the earliest bacterial node (Figure 8). This suggests that adaptation to growth on acetate took place once in bacteria, and that the essential replacements have been preserved in the various diverse bacterial lineages for 3.5 billion years. Interestingly, none of these replacements is found in the cytoplasmic NADP-dependent ICDHs, where a large conserved insert, slap bang in the middle of loop 3 of the coenzyme-binding pocket (Figure 1), strongly suggests that an alternative means to bind NADP evolved independently in eukaryotes.
In contrast to the conservation seen in bacterial NADP-dependent ICDHs, only one of the five amino acids is conserved in all related NAD-dependent enzymes. This is the aspartate (344), so crucial to hydrogen-bond formation with the hydroxyls on the ribose-sugar portion of NAD. Alanine (351) and isoleucine (345) are occasionally replaced by other residues retaining two essential characteristics—that they be small and hydrophobic. A veritable riot of replacements can be found at sites 292' and 391, no doubt because these sites are no longer of any functional significance whatsoever to NAD binding.
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