Crystal structure of spermidine synthase fromHelicobacter pylori

Abstract

binding channel shows conservation of positively charged residues, which are possibly involved in complex formation with the H-protein. The structure of I c consists of a major -type domain and an Nterminal helical segment. I c has two CPY-binding sites: the N-terminal inhibitory reactive site and the secondary CPY-binding site which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. I c also has the ligand-binding site, the putative binding site of the polar head group of phospholipid, which is conserved among PEBPs and accommodates a sulfate ion in the crystal structure.Along with the complex structure of I c , its mutational analyses for inhibitory activity and binding to CPY demonstrate that the Nterminal inhibitory reactive site is essential for the complex formation with CPY as well as enzyme inhibition and that the I c binding to CPY forms a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of Ic toward CPY gives insights into not only the inhibitory mechanism of PEBPs toward serine proteinases but also the biological functions of I c belonging to the PEBP family.[1] Mima J., Hayashida M., Fujii T., Hata Y., Hayashi R., Ueda M. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine in the biosynthesis of spermidine. PAPT of Helicobacter pylori (HpPAPT) is encoded by the speE gene. HpPAPT has less than 20% of sequence identity with other PAPTs, even containing no signature sequence. The threedimensional structure of HpPAPT has been determined by multiwavelength anomalous dispersion (MAD) in this study. HpPAPT consists of an N-terminal beta-stranded domain and a Cterminal Rossmann-like domain, with a binding pocket between two domains. The oligomerization of HpPAPT is mostly made by the Nterminal domain and sensitive to the pH values of buffer. Our structure illustrates that HpPAPT has a distinctive binding pocket with a bigger space, a unique electrostatic potential surface of less acidity, and numerous unconserved residues. Due to the lack of the gatekeeping loop, HpPAPT may need to perform a significant conformation change to accommodate the ligand binding. When the tryptophan synthase -and 2 -subunits combine to form the 2 2 complex, the enzymatic activity of each subunit is stimulated by one to two orders of magnitude. In order to elucidate the structural basis of this mutual activation, it is necessary to determine the structures of the -and -subunits alone and together with the 2 2 complex. The crystal structures of the tryptophan synthase 2 2 complex from S. typhimurium (St 2 2 ) has been reported. Therefore, we determined the crystal structure of the tryptophan synthasesubunit alone from E. coli (Ec ) at 2.3Å resolution. The biggest difference between the structures of the Ec and the -subunit in the St 2 2 (St ) was as follows. The helix-2' in the St including an ...