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Acta Crystallographica Section D: Biological Crystallography (1)
Journal of Virology (1)
Attrill, Helen (1)
Attrill, Helen L. (1)
Blair, David E. (1)
Clements, J. Barklie (1)
Cumming, Sarah A. (1)
Dorfmueller, Helge C. (1)
Fang, Wenxia (1)
Graham, Sheila V. (1)
Rao, Francesco V. (1)
van Aalten, Daan M. F. (1)
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Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1
Dorfmueller, Helge C.
Rao, Francesco V.
Blair, David E.
van Aalten, Daan M. F.
Acta Crystallographica Section D: Biological Crystallography
Glucosamine-6-phosphate N-acetyltransferase is an essential enzyme of the eukaryotic UDP-GlcNAc biosynthetic pathway. A crystal structure at 1.55 Å resolution revealed a highly unusual covalent product complex and biochemical studies investigated the function of a fully conserved active-site cysteine.
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.
carbohydrates; glycobiology; Caenorhabditis elegans; glucosamine-6-phosphate N-acetyltransferase; coenzyme A adduct; mechanism
The Herpes Simplex Virus Type 1 US11 Protein Binds the Coterminal UL12, UL13, and UL14 RNAs and Regulates UL13 Expression In Vivo
Cumming, Sarah A.
Clements, J. Barklie
Graham, Sheila V.
Journal of Virology
The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. US11 localizes to the nucleolus in infected cells, can associate with ribosomes, and has been shown to bind RNA. The RNA substrates of US11 identified thus far have no apparent role in the virus lytic cycle, so we set out to identify a novel, biologically relevant RNA substrate(s) for this protein in HSV-1-infected cells. We designed a reverse transcriptase PCR-based protocol that allowed specific selection of a 600-bp RNA binding partner for US11. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. We show that the binding of US11 to 12/14 is sequence-specific and mediated by the C-terminal domain of the protein. To elucidate the role of US11 in the virus life cycle, we infected cells with wild-type virus, a cosmid-reconstructed US11 HSV-1 null mutant, and a cosmid-reconstructed wild-type virus and analyzed expression of UL12, -13, and -14 during a time course of infection. These experiments revealed that this interaction has biological activity; at early times of infection, US11 down-regulates UL13 protein kinase mRNA and protein.
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