DyP, a unique dye-decolorizing enzyme from the fungus Thanatephorus cucumeris Dec 1, has been classified as a peroxidase but lacks homology to almost all other known plant peroxidases. The primary structure of DyP shows moderate sequence homology to only two known proteins: the peroxidedependent phenol oxidase, TAP, and the hypothetical peroxidase, cpop21. Here, we show the first crystal structure of DyP and reveal that this protein has a unique tertiary structure with a distal heme region that differs from that of most other peroxidases. DyP lacks an important histidine residue known to assist in the formation of a Fe 4؉ oxoferryl center and a porphyrinbased cation radical intermediate (compound I) during the action of ubiquitous peroxidases. Instead, our tertiary structural and spectrophotometric analyses of DyP suggest that an aspartic acid and an arginine are involved in the formation of compound I. Sequence analysis reveals that the important aspartic acid and arginine mentioned above and histidine of the heme ligand are conserved among DyP, TAP, and cpop21, and structural and phylogenetic analyses confirmed that these three enzymes do not belong to any other families of peroxidase. These findings, which strongly suggest that DyP is a representative heme peroxidase from a novel family, should facilitate the identification of additional new family members and accelerate the classification of this novel peroxidase family.Peroxidases have been systematically researched for more than 70 years. Fifteen years ago, Welinder (1) proposed the concept of a "plant peroxidase superfamily" comprising classes I, II, and III, based on primary sequence alignments and isolation from prokaryotes, fungi, and plants, respectively. Using this strategy, yeast cytochrome c peroxidase (2) and chloroplast ascorbate peroxidase (3) were classified as class I peroxidases on account of their prokaryotic source. Representatives of class II include lignin peroxidase (LiP) 2 (4), manganese peroxidase (MnP) (5), and versatile peroxidase (6, 7), whereas class III contains horseradish peroxidase (HRP) (8) and barley grain peroxidase (BGP) (9). This classification has been widely applied to most known peroxidases, with the exception of chloroperoxidase (CPO) isolated from the fungus, Caldariomyces fumago, which lacks primary structural homology with other peroxidases (10, 11). However, in contrast to the plant peroxidases, those from animals, including mammals, are yet to be categorized. To date, most of these enzymes have been grouped into the plant or animal peroxidase superfamily (12).So far, we isolated and characterized a novel extracellular peroxidase, DyP, from the fungus Thanatephorus cucumeris Dec 1 (13-16). DyP, a glycoprotein having one heme as a cofactor, has a molecular mass of 58 kDa and requires H 2 O 2 for all enzyme reactions, indicating that it functions as a peroxidase. DyP has several characteristics that distinguish it from all other peroxidases, including a particularly wide substrate specificity, a lack of homology to m...

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50 degrees C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50 degrees C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55-60 kDa, as determined by SDS-PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.

The growth of suitably sized protein crystals is essential for protein structure determination by X-ray crystallography. In general, crystals are grown using a trial-and-error method. However, these methods have been modified with the advent of microlitre dispensing-robot technology and of protocols that rapidly screen for crystal nucleation conditions. The use of one such automatic dispenser for mixing protein drops (1.3-2.0 microl in volume) of known concentration and pH with precipitating solutions (ejecting 2.0 microl droplets) containing salt is described here. The results of the experiments are relevant to a crystallization approach based on a two-step procedure: screening for the crystal nucleation step employing robotics followed by optimization of the crystallization conditions using incomplete factorial experimental design. Large crystals have successfully been obtained using quantities as small as 3.52 mg protein.

No abstract