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
Protein Engineering
That only six out of approximately 250 amino acid replacements at 320 positions (excluding all gaps) separating your typical bacterial NADP-dependent ICDH from your typical NAD-dependent enzyme are directly involved with determining coenzyme usage might seem reasonable to a protein chemist. However, it seems downright absurd to most evolutionary biologists brought up on a steady diet of pan-gradualism: many mutations, each of small effect, each improving the phenotype—just a little bit. Clearly, an experiment is in order.
The experiment is conceptually simple. The key amino acid residues of an NAD-dependent enzyme are introduced into E. coli NADP-dependent ICDH (which, recall, prefers to use NADP) using sequential rounds of site-directed mutagenesis, a technique of molecular biology whereby the amino acid sequence of a protein can be changed by introducing specific changes into the gene encoding it. The engineered gene then directs the synthesis of the engineered enzyme, which is then purified and its properties are studied.
Assuming the protein chemists are correct, the engineered proteins should change their coenzyme usage. Engineered ICDH, with the proper six amino acid replacements, should display a marked preference for NAD. Similarly, an engineered IPMDH should display a marked preference for NADP (wild-type IPMDH prefers to use NAD). A failure to switch coenzyme usage in either enzyme would indicate that additional, as yet unidentified, residues make significant contributions to coenzyme preference. Success in both cases would indicate that the key amino acid residues determining coenzyme preference have been correctly identified and that all remaining amino acid replacements separating NAD users from NADP users are of little or no significance with regard to this phenotype.
To determine the success f the experiment, we need a measure of the ability of an enzyme to discriminate between the two coenzymes. To do this we compare how efficiently each enzyme uses one coenzyme compared with the other. For example, it can be shown that wild-type E. coli ICDH uses 6,800 molecules of NADP for every molecule of NAD used, when both coenzymes are present in equal concentrations.
Five amino acid replacements in the coenzyme-binding pocket of E. coli ICDH shift preference away from NADP toward NAD by a factor of 106. However, the overall catalytic efficiency is far lower than that of a protein that evolved naturally to use NAD, the yeast mitochondrial NAD-dependent ICDH. This is not altogether surprising. The changes introduced to the coenzyme binding-pocket are quite drastic—whole chemical groups have been added, others have been eliminated, and the charge surrounding the pocket has been changed by four units. Optimizing activity may often require changes of a more subtle nature: an atom to be nudged a fraction over here, the angle of a hydrogen bond to be changed by 15 degrees over there, a minor steric clash to be eliminated. Such subtle changes can only be introduced by replacing amino acids at some distance from the coenzyme-binding pocket in the hope that their gross local effects diminish with distance. We tested this hypothesis by introducing bulky amino acids, to force conformational changes, at six sites surrounding the active site of ICDH. Two mutants combine to increase catalytic efficiency by a factor of 16, bringing it in line with that of the yeast enzyme (Figure 11).
Both the engineered E. coli ICDH and yeast mitochondrial ICDH have lower overall catalytic efficiencies than wild-type E. coli ICDH. This is because five hydrogen bonds and three charge interactions allow NADP to bind more tightly than NAD, which instead is stabilized by a double hydrogen bond. On the other hand, the engineered ICDH has a higher specificity for its new coenzyme than does the naturally evolved IPMDH. Moreover, x-ray analysis reveals that NAD occupies precisely the same position in engineered ICDH that it does in wild-type IPMDH. By any measure, a highly specific NAD-dependent ICDH has been engineered. The last time such an enzyme used NAD in preference to NADP was 3.5 billion years ago!
Well, if you can engineer one way, you should be able to engineer the other way as well. However, engineering the coenzyme specificity of T. thermophilus IPMDH is far trickier than engineering ICDH. Now we must engineer the very architecture of a protein. The sharp turn of IPMDH must be replaced by a helix and loop so as to introduce an additional arginine residue, (395), to form a hydrogen bond with the 2'-phosphate of NADP (Figure 10). A 13-residue sequence modeled on the helix and loop of E. coli ICDH, but containing additional amino acid replacements to ensure that the insert lies correctly against the remaining protein, replaced the seven residues comprising the sharp turn in IPMDH.
Together with four direct replacements lining the pocket (sites 292', 344, 345 and 351), a shift in preference from NAD to NADP by a factor of 100,000 was generated. The resulting mutant IPMDH contains the entire suite of amino acids found in ICDH, has a specificity for NADP of 1,000 and is twice as active as the wild-type enzyme (Figure 11). A highly specific NADP-dependent IPMDH has been engineered. The last time an IPMDH used NADP in preference to NAD was, well, never. All available evidence is consistent with the hypothesis that this engineered phenotype is unique in the entire history of life.
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