Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O(gamma) of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.
The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADPglucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33°C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only ;50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.
Previous studies have shown a statistically significant correlation between human carcinomas and monoclonal antibody detection of a Mycoplasma hyorhinis -encoded protein known as p37. A potential mechanism of p37 is that it might promote invasion and metastasis. Recombinant p37 enhanced the invasiveness of two prostate carcinoma and two melanoma cell lines in a dosedependent manner in vitro, but did not have a significant effect on tumor cell growth. Furthermore, the increased binding to cell surfaces and the enhanced invasive potential of cancer cells from exposure to p37 could be completely reversed by preincubation of the cancer cells with an anti-p37 monoclonal antibody. Sequence comparisons, followed by three-dimensional molecular modeling, revealed a region of similarity between p37 and influenza hemagglutinin A, a sialic acid -binding protein that plays a critical role in viral entry. Binding of p37 to prostate carcinoma cells was found to be at least partially sialic acid dependent because neuraminidase treatment decreased this binding. Taken together, these observations suggest that M. hyorhinis can infect humans and may facilitate tumor invasiveness via p37. These results further suggest that p37 may be a molecular target for cancer therapy. [Mol Cancer Ther 2005;4(7):1031 -8]
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