A journey back in time is possible at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx), dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32° C more stable than modern enzymes and the oldest show significantly higher activity than extant ones at pH 5. We probed their mechanisms of reduction using single-molecule force spectroscopy. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs utilize chemical mechanisms of reduction similar to those of modern enzymes. While Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over a 4 Gyr time span to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.
Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.
The geometric structure of the catalytically relevant ferrous active site of phenylalanine hydroxylase (PAH) has been investigated using magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies. From the excited state ligand field transitions in the MCD spectrum (10Dq ) 9400 cm -1 , ∆ 5 E g ) 1800 cm -1 ), the temperature and field dependence of these transitions (∆ 5 T 2g ) 590 cm -1 ), and the XAS pre-edge shapes and intensities, the resting ferrous site of the "tense" form of PAH is six-coordinate distorted octahedral. The low ligand field strength observed in the MCD spectrum results from significant oxygen ligation and longer Fe-O/N bond distances relative to model complexes as determined from an EXAFS analysis. Nonallosteric activation using N-ethylmaleimide does not notably affect the band positions in the MCD spectrum and therefore does not perturb the structure of the iron center. However, substrate addition without allosteric activation results in a different six-coordinate distorted octahedral structure as determined by MCD (10Dq ) 10 000 cm -1 , ∆ 5 E g ) 1450 cm -1 ) and XAS with a greater d π -orbital splitting (∆ 5 T 2g ) 1050 cm -1 ). EXAFS analysis indicates a shift in the relative number of ligands from the outer to the inner subshell of the first coordination sphere in the substrate-bound form of the enzyme relative to the resting site, consistent with the increased ligand field strength observed directly from the MCD spectrum. Substrate-induced allosteric activation (∼34 kcal/mol) does not alter the structure of the iron site in the "relaxed" form of PAH compared to the substrate-bound "tense" state. Thus, while activation is necessary for the enzyme to achieve complete catalytic competence, it does not appear to affect the geometry of the catalytically relevant six-coordinate ferrous active site and only directly influences the surrounding protein conformation. In contrast, substrate addition results in a geometric and electronic structural change at the iron center which may help orient the substrate for completely coupled hydroxylation.
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