In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations. The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.

In certain maize genotypes, called "null," ␤-glucosidase does not enter gels and therefore cannot be detected on zymograms after electrophoresis. Such genotypes were originally thought to be homozygous for a null allele at the glu1 gene and thus devoid of enzyme. We have shown that a ␤-glucosidase-aggregating factor (BGAF) is responsible for the "null" phenotype. BGAF is a chimeric protein consisting of two distinct domains: the disease response or "dirigent" domain and the jacalin-related lectin (JRL) domain. First, it was not known whether the lectin domain in BGAF is functional. Second, it was not known which of the two BGAF domains is involved in ␤-glucosidase binding and aggregation. To this end, we purified BGAF to homogeneity from a maize null inbred line called H95. The purified protein gave a single band on SDS-PAGE, and the native protein was a homodimer of 32-kDa monomers. Native and recombinant BGAF (produced in Escherichia coli) agglutinated rabbit erythrocytes, and various carbohydrates and glycoproteins inhibited their hemagglutination activity. Sugars did not have any effect on the binding of BGAF to the ␤-glucosidase isozyme 1 (Glu1), and the BGAF-Glu1 complex could still bind lactosyl-agarose, indicating that the sugar-binding site of BGAF is distinct from the ␤-glucosidase-binding site. Neither the dirigent nor the JRL domains alone (produced separately in E. coli) produced aggregates of Glu1 based on results from pull-down assays. However, gel shift and competitive binding assays indicated that the JRL domain binds ␤-glucosidase without causing it to aggregate. These results with those from deletion mutagenesis and replacement of the JRL domain of a BGAF homolog from sorghum, which does not bind Glu1, with that from maize allowed us to conclude that the JRL domain of BGAF is responsible for its lectin and ␤-glucosidase binding and aggregating activities.␤-Glucosidases (␤-D-glucoside glucohydrolase; EC 3.2.1.21) hydrolyze ␤-glycosidic linkage(s) in alkyl and aryl ␤-D-glucosides, glycoproteins, and glycolipids and that between two glucose residues in ␤-linked oligosaccharides. They are found in all three (Archaea, Eubacteria, and Eukarya) domains of liv-

SUMMARY The antimycobacterial efficacy of the abyssomicin C family of natural products, in addition to a key synthetic intermediate, has been investigated given their reported inhibition of Bacillus subtilis p-aminobenzoate biosynthesis. The naturally occurring (−)-abyssomicin C and its atropisomer were found to exhibit low micromolar growth inhibition against the relatively fast-growing and non-virulent Mycobacterium smegmatis and the vaccine strain Mycobacterium bovis BCG, while their antipodes were slightly less active. (−)-Abyssomicin C and its atropisomer were particularly efficacious against Mycobacterium tuberculosis H37Rv, exhibiting MIC values of 3.6 and 7.2 μM, respectively. More specifically, (−)-abyssomicin C was bactericidal. This complex natural product and its analogs, thus, hold promise as chemical tools in the study of M. tuberculosis metabolism.

Background: Polyketide synthases (PKSs) synthesize complex lipid components of the cell wall in M. tuberculosis. Results: We determined the crystal structure of M. tuberculosis PKS11 and identified its substrate, intermediates, and product. Conclusion: M. tuberculosis PKS11 synthesizes a unique cyclic methyl-branched alkylpyrone. Significance: Our identification of alkylpyrones as the product of this PKS suggests a previously unknown component of the mycobacterial cell wall.

Asialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPOP). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPOP was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep-tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPOP expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPOP revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC-MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPOP. However, Strep-tag II was detected on asialo-rhuEPOP that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.