and Cerasoli, Douglas M., "Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPaselike homology model" (2004 AbstractHuman serum paraoxonase (HuPON1) is a calcium-dependent enzyme that hydrolyzes esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzyme's arylesterase and organophosphatase activities. Until very recently, a three-dimensional model was not available for HuPON1, so functional roles have not been assigned to those residues. Based on sequence-structure alignment studies, we have folded the amino acid sequence of HuPON1 onto the sixfold h-propeller structure of squid diisopropylfluorophosphatase (DFPase). We tested the validity of this homology model by circular dichroism (CD) spectroscopy and site-directed mutagenesis. Consistent with predictions from the homology model, CD data indicated that the structural composition of purified HuPON1 consists mainly of h-sheets. Mutants of HuPON1 were assayed for enzymatic activity against phenyl acetate and paraoxon. Substitution of residues predicted to be important for substrate binding (L69, H134, F222, and C284), calcium ion coordination (D54, N168, N224, and D269), and catalytic mechanism of HuPON1 (H285) led to enzyme inactivation. Mutants F222Y and H115W exhibited substrate-binding selectivity towards phenyl acetate and paraoxon, respectively. The homology model presented here is very similar to the recently obtained PON1 crystal structure, and has allowed identification of several residues within the enzyme active site. D

Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities.

Peroxiredoxin VI (Prdx6) is an antioxidant enzyme highly expressed in the lungs. Its antioxidant properties are due to its ability to reduce hydroperoxides found in lung surfactants and thus prevent the toxicity associated with hyperoxia. Prdx6 is a bifunctional protein that contains two distinct active sites. One active site catalyzes a phospholipase A2 (PLA2) type hydrolysis of phospholipids, and the second active site catalyzes the reduction of lipid hydroperoxides typical of 1‐cys peroxiredoxins. The structure of Prdx6 has been solved; however, there is no structural evidence to elucidate its catalytic mechanism. This study is designed to solve the structure of Prdx6 in complex with MJ33, a PLA2 transition‐state inhibitor, in an effort to understand the mechanism and conformational change Prdx6 undergoes during its catalytic cycle. Here we report that Prdx6 has been successfully purified to homogeneity. Our crystallization screens have produced protein crystals that have diffracted to a resolution of 2.8Å. This research is supported by the Chemistry Alumni Scholars.