N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide-activated UDPGlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 Å resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long α-helical arm: (i) an N-terminal domain which resembles the dinucleotidebinding Rossmann fold; and (ii) a C-terminal domain which adopts a left-handed parallel β-helix structure (LβH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LβH domains, the long α-helical linkers being seated on top of the arrangement and the N-terminal domains projected away from the 3-fold axis. In addition, the 2.3 Å resolution structure of the GlmU-UDP-GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three-dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP-sugar bacterial pyrophosphorylases.

Self-incompatibility (SI) inBrassica species is controlled by a single polymorphic locus ( S ) with multiple specificities. Two stigmatically expressed genes that have been cloned from this region encode the S locus glycoprotein (SLG) and S receptor kinase (SRK). Both appear to be essential for the operation of SI. It is believed that rejection of incompatible pollen grains is effected by recognition events between an as yet unidentified S locus-encoded pollen coating-borne protein and the SLG/SRK. We previously identified a small pollen coat protein PCP7 (renamed here PCP-A1, for pollen coat protein, class A, 1) that binds with high affinity to SLGs irrespective of S genotype. Here, we report the cloning of PCP-A1 from Brassica oleracea and demonstrate that it is unlinked to the S locus. In situ localization of PCP-A1 transcripts revealed that they accumulate specifically in pollen at the late binucleate/trinucleate stage of development rather than in the tapetum, which previously was taken to be the principal source of the pollen coat. PCP-A1 is characterized by the presence of a structurally important motif consisting of eight cysteine residues shared by the plant defensins. Based on the presence of this motif and other data, homology modeling has been used to produce a putative structure for PCP-A1. Protein-protein interaction analyses demonstrate that SLG exists in monomeric and dimeric forms, both of which bind PCP-A1. Evidence is also presented for the existence of putative membrane-associated PCP-A1 binding proteins in stigmatic tissue. INTRODUCTIONFlowering plants have evolved a range of both morphological and molecular mechanisms that prevent self-pollination. It has been estimated that up to 50% of angiosperms possess such self-incompatibility (SI) systems (Darlington and Mather, 1949; Brewbaker, 1959), highlighting their evolutionary importance (Stebbins, 1950(Stebbins, , 1957 Whitehouse, 1950;de Nettancourt, 1977). The most advanced SI systems are thought to be those that are controlled by a single multiallelic locus ( S locus), typified by Brassica , Nicotiana , and Papaver spp. In Nicotiana and Papaver spp, in which pollen compatibility is controlled by the gametophyte, the grain is rejected if the S allele it carries is shared by the stigma. Importantly, pollen compatibility in Brassica spp is regulated by the sporophyte, and thus the S alleles of the pollen parent determine whether the grain is accepted or rejected. In recent years, advances have been made toward elucidating the molecular basis of SI in these and other species (reviewed in Franklin et al., 1995;Hiscock et al., 1996;Kao and McCubbin, 1996).The Brassica S locus has been estimated to span several hundred kilobases of DNA (Boyes and Nasrallah, 1993). To date, two polymorphic stigmatically expressed S locus genes that appear to be required for the operation of SI have been cloned; one encodes a transmembrane receptor-like protein kinase (Stein et al., 1991) termed SRK (for S receptor kinase), and the other encodes a glycoprotein (Na...

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea. In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an ‘extension’ of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of ‘self’ coating (i.e. that with the same S‐haplotype as the stigma) prevent the success of a compatible cross pollination, but a ‘cross’ coating (i.e. that with a different S‐haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen‐held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an Mr10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine‐rich proteins (PCP‐A: Pollen Coat Proteins‐class A)—one of which is known to bind to stigmatically‐expressed components of the S‐locus in Brassica. Introduction of the PCP‐A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP‐A protein family.