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Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells.Here we characterize the sugar-binding specificity of Siglec-7 expressed on Chinese hamster ovary cells using polyvalent streptavidin-based glyco-probes. Glycoprobes carrying unique oligosaccharide structures such as GD3 (NeuAc␣2,8NeuAc␣2,3Gal␤1,4Glc) and LSTb (Gal␤1,3[NeuAc␣2,6]GlcNAc␤1,3Gal␤1,4Glc) oligosaccharides bound to Siglec-7 better than those carrying LSTc (NeuAc␣2,6Gal␤1,4GlcNAc␤1,3Gal␤1,4Glc) or GD1a (NeuAc␣2,3Gal␤1,3GalNAc␤1,4[NeuAc␣2,3]Gal-␤1,4Glc) oligosaccharides. In contrast, Siglec-9, which is 84% identical to Siglec-7, did not bind to the GD3 and LSTb probes but did bind to the LSTc and GD1a probes. To identify a region(s) responsible for their difference in binding specificity, we prepared a series of V-set domain chimeras between Siglecs-7 and -9. Substitution of a small region, Asn 70 -Lys 75 , of Siglec-7 with the equivalent region of Siglec-9 resulted in loss of Siglec-7-like binding specificity and acquisition of Siglec-9-like binding properties. In comparison, a Siglec-9-based chimera, which contains Asn 70 -Lys 75 with additional amino acids derived from Siglec-7, exhibited Siglec-7-like specificity. These results, combined with molecular modeling, suggest that the C-C loop in the sugar-binding domain plays a major role in determining the binding specificities of Siglecs-7 and -9.

ATP-phosphoribosyltransferase (ATPPRT) catalyses the first step in histidine biosynthesis, the condensation of ATP and 5-phospho--D-ribosyl-1-pyrophosphate to generate N 1 -(5-phospho--D-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain, and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was solved at 2.34-Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-to 4-fold increase in kcat.Reaction profiles for both enzymes are indistinguishable by 31 P-NMR, indicating that the same reaction is catalysed. Temperature dependence of kcat shows deviation from Arrhenius behaviour at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 K and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterisation of a psychrophilic ATPPRT. 4Adenosine 5ʹ-triphosphate phosphoribosyltransferase (ATPPRT) (EC 2.4.2.17) catalyses the reversible Mg 2+ -dependent reaction between adenosine 5ʹ-triphosphate (ATP) and(PR-ATP) and inorganic pyrophosphate (PPi) (Scheme 1), the first step in histidine biosynthesis. 1 The chemical equilibrium of the reaction strongly favours reactants, 2 and the enzyme is allosterically inhibited by histidine. 1 In addition to being a model for understanding allostery, 2-4 ATPPRT is of biotechnological interest as a tool for histidine production, provided that histidine feedback inhibition can be overcome. [5][6][7] Two forms of ATPPRT, encoded by the hisG gene, are found in nature. Fungi, plants, and most bacteria possess a long, homo-hexameric protein, HisGL, each subunit consisting of two domains that make up the catalytic core and a C-terminal regulatory domain that mediates feedback inhibition by histidine. 8 Some bacteria and archaea have a short version of the protein, HisGS, which lacks the C-terminal regulatory domain and is insensitive to histidine. In these organisms, a catalytically inactive regulatory protein, HisZ, the product of the hisZ gene, is present. 9 HisZ, which shares a common ancestry with histidyl-tRNA synthetase (HisRS), binds HisGS to form ...