Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys 73 and Cys 120 in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4 -5B are highly ␤-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to ␤-strands, thereby forming sequential intermolecular linkages.The aggregation of misfolded proteins is of significant importance in biology because this phenomenon is closely related to numerous human diseases, including neurodegenerative diseases, such as Alzheimer and Parkinson diseases (1, 2). Amyloid fibril formation of denatured proteins and polymerization of the serpin family of serine protease inhibitors provide well defined structural models of neurodegenerative diseases. The serpins possess a metastable conformation that can undergo a unique conformational change involving the insertion of a proteolytically cleaved reactive center loop into the central ␤-sheets (3, 4), which is required for their inhibitory function. Mutations of serpins lead to their polymerization and intracellular aggregation. Polymer formation of serpins, such as ␣ 1 -antitrypsin, is associated with liver cirrhosis (5), whereas that of neuroserpin (6) results in a cumulative loss of neurons and the onset of Alzheimer-like dementia.The mechanism of serpin polymerization is currently being investigated (7-10). The best described case is provided by the polymerization of the Z variant of ␣ 1 -antitrypsin (11); the Z mutation has a minimal effect on the activity or stability of the native protein (10), and the defect in secretion is due to a perturbation of the folding pathway. A recent structural study of antithrombi...

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The directed evolution of the thermotolerant NADP(H)-dependent malic enzyme from Thermococcus kodakarensis was conducted to alter the cofactor preference of the enzyme from NADP(H) to NAD(H). The construction and screening of two generations of mutant libraries led to the isolation of a triple mutant that exhibited 6-fold higher kcat/Km with NAD(+) than the wild type. We serendipitously found that, in addition to the change in the cofactor preference, the reaction specificity of the mutant enzyme was altered. The reductive carboxylation of pyruvate to malate catalyzed by the wild type enzyme is accompanied by HCO(3)(-)-independent reduction of pyruvate and gives lactate as a byproduct. The reaction specificity of the triple mutant was significantly shifted to malate production and the mutant gave a less amount of the byproduct than the wild type. When the triple mutant enzyme was used as a catalyst for pyruvate carboxylation with NADH, the enzyme gave 1.2 times higher concentration of malate than the wild type with NADPH. Single-point mutation analysis revealed that the substitution of Arg221 with Gly is responsible for the shift in reaction specificity. This finding may shed light on the catalytic mechanisms of malic enzymes and other related CO2- and/or HCO(3)(-)-fixing enzymes.

Plasma and erythrocyte enzyme activities were measured in ddY mice supplemented with dietary selenium (Se) from baker's yeast (Saccharomyces serevisiae) or selenious acid at 0.3 ppm Se content. Glutathione peroxidase (GSHpx) activities increased significantly in erythrocytes from mice supplemented with dietary Se. It was concluded that addition of dietary Se as food additive is very effective for activation of GSHpx in mice.

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