Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.

uses a variant Bt Cry3Bb1 insecticidal protein (Donovan et al., 1992). Cry3Bb1 is known to be biologically active The corn rootworm (CRW; Diabrotica spp.) is one of the most against several species within the Coleopteran family serious pests of corn in the USA. Chemical insecticides and crop rotation have been the only two options available to growers for Chrysomelidae, including the western corn rootworm, managing CRW. Unfortunately, both of these tactics can be ineffective Diabrotica virgifera virgifera LeConte (Rupar et al., as a result of either resistance or behavioral modifications. In this 1991). The biological activity of this protein against D. paper, we describe transgenic maize (Zea mays L.) hybrids that control virgifera virgifera suggested its potential use in creating CRW. These hybrids were created with a Cry3Bb1 Bacillus thurintransgenic plants expressing Cry3Bb1 that would confer giensis (Bt) variant that is approximately eight times more lethal to protection to corn root tissue from larval feeding damcorn rootworm larvae than the wild-type protein. A DNA vector age. To further augment protection of the root system containing the modified cry3Bb1 gene was placed under control of a from larval feeding damage, modifications were introroot-enhanced promoter (4-AS1) and was introduced into embryonic duced in the cry3Bb1 gene that gave rise to an amino acid maize cells by microprojectile bombardment. Described here is the variant Cry3Bb1 protein with an eight-fold increase in molecular genetic characterization, protein expression levels, and field performance of the recently commercialized MON863 hybrids.

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Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among t h e subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, t h e M, 160,000 subunit Vt is cleaved to products of M, 95,000 and M, 50,000. In association with an unprocessed M, 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 3OoC), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose-type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4-5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event.Although the predominant oligosaccharide of Vt is Man9GlcNAc2, the M, 50,000 subunit of egg-borne Vt contains a much higher proportion of Man6GlcNAc2 than t h e other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an ootheca permits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo-a-rnannosidase, exo-0-Nacetylgl ucosam i n idase, a-glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo-P-N-acetyl- glucosarninidase activities coincide with the day 4-5 proteolysis, while arnannosidase remains relatively constant throughout the first 6 days.Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.