			Home About us Contact

Putative Active Site (putative + active_site)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

Structure of serine acetyltransferase from Haemophilus influenzae Rd

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004
Jason Gorman
The crystal structure of serine acetyltransferase (SAT) from Haemophilus influenzae Rd determined at 2.7,Å resolution is presented. SAT is a member of a family of hexapeptide-containing transferases that contain six-residue tandem repeats (LIV)-G- X4 that have been shown to form left-handed parallel ,-helices. In the current structure, each protomer is comprised of two domains: an N-terminal ,-helical domain and a C-terminal left-handed parallel ,-helix domain. Although other members of this protein family are known to form trimeric structures, SAT forms a dimer of trimers in which the trimer interface is mediated through interactions between both the ,-helix domains and N-terminal domains; these trimers dimerize through contacts in the N-terminal domain. All dimer-of-trimer interactions are mediated through amino acids within an N-terminal extension common only to a subset of SATs, suggesting that members of this subfamily may also adopt hexameric structures. Putative active sites are formed by crevices between adjacent protomers in a trimer. Thus, six independent active sites exist in the hexameric enzyme complex. [source]

Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase

FEBS JOURNAL, Issue 24 2000
Juan C. Díaz Arribas
Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2,,4,-dinitrophenyl 2-deoxy-2-fluoro-,- d -glucopyranoside (1) and 2,,4,-dinitrophenyl-2-deoxy-2-fluoro-,- d -galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (d -glucal and d -galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett.435, 225,228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem.267, 18744,18752]. [source]

Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S -adenosyl- l -homocysteine

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005
Augen A. Pioszak
The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S -adenosyl- l -methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S -adenosyl- l -homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55,Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60,Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central ,/,-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA. [source]

Crystal structures of possible lysine decarboxylases from Thermus thermophilus HB8

PROTEIN SCIENCE, Issue 11 2004
Mutsuko Kukimoto-Niino
Abstract TT1887 and TT1465 from Thermus thermophilus HB8 are conserved hypothetical proteins, and are annotated as possible lysine decarboxylases in the Pfam database. Here we report the crystal structures of TT1887 and TT1465 at 1.8 Å and 2.2 Å resolutions, respectively, as determined by the multiwavelength anomalous dispersion (MAD) method. TT1887 is a homotetramer, while TT1465 is a homohexamer in the crystal and in solution. The structures of the TT1887 and TT1465 monomers contain single domains with the Rossmann fold, comprising six , helices and seven , strands, and are quite similar to each other. The major structural differences exist in the N terminus of TT1465, where there are two additional , helices. A comparison of the structures revealed the elements that are responsible for the different oligomerization modes. The distributions of the electrostatic potential on the solvent-accessible surfaces suggested putative active sites. [source]