title: Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase.
abstract: Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.
authors.affiliation: Unit of Immunology and General Pathology, Department of Molecular Medicine, University of Pavia, Via Ferrata 9, Pavia 27100, Italy. giovanna.valentini@unipv.it.|Laboratory of Protein Biochemistry, Department of Biology and Biotechnologies "L. Spallanzani", University of Pavia, Viale Taramelli 3/B, Pavia 27100, Italy. giovanna.valentini@unipv.it.
authors.forename: Maristella
authors.initials: M
authors.lastname: Maggi
authors.affiliation: Laboratory of Molecular Microbiology, Department of Biology and Biotechnologies "L. Spallanzani", University of Pavia, Via Ferrata 9, Pavia 27100, Italy. laurent.chiarelli@unipv.it.
authors.forename: Laurent R
authors.initials: LR
authors.lastname: Chiarelli
authors.affiliation: Laboratory of Protein Biochemistry, Department of Biology and Biotechnologies "L. Spallanzani", University of Pavia, Viale Taramelli 3/B, Pavia 27100, Italy. giovanna.valentini@unipv.it.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Valentini
authors.affiliation: Unit of Immunology and General Pathology, Department of Molecular Medicine, University of Pavia, Via Ferrata 9, Pavia 27100, Italy. claudia.scotti@unipv.it.
authors.forename: Claudia
authors.initials: C
authors.lastname: Scotti
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004795:Enzyme Stability; D016480:Helicobacter pylori / Q000201:enzymology*; D008969:Molecular Sequence Data
chemical_list: D001426:Bacterial Proteins; D001215:Asparaginase
references.citation: J Mol Biol. 2007 Jun 8;369(3):794-811
references.pmid: 17451745
references.citation: Biochem Biophys Res Commun. 2008 Dec 26;377(4):1222-6
references.pmid: 18983825
references.citation: Recent Pat Anticancer Drug Discov. 2012 Jan;7(1):4-13
references.pmid: 21854356
references.citation: Protein Expr Purif. 2005 May;41(1):207-34
references.pmid: 15915565
references.citation: Acta Crystallogr D Biol Crystallogr. 2009 Dec;65(Pt 12):1253-61
references.pmid: 19966411
references.citation: Biochim Biophys Acta. 2000 Sep 29;1481(2):349-59
references.pmid: 11018727
references.citation: Trends Biochem Sci. 1998 Sep;23(9):347-52
references.pmid: 9787641
references.citation: Acta Biochim Pol. 2006;53(4):627-40
references.pmid: 17143335
references.citation: PLoS One. 2015;10(2):e0117025
references.pmid: 25664771
references.citation: PLoS One. 2010;5(11):e13892
references.pmid: 21085483
references.citation: Biochim Biophys Acta. 1975 Apr 29;386(2):576-89
references.pmid: 237538
references.citation: Enzyme Microb Technol. 2001 May 7;28(7-8):696-704
references.pmid: 11339955
references.citation: Methods Enzymol. 1985;113:608-18
references.pmid: 3911011
references.citation: Biochemistry. 2001 May 15;40(19):5655-64
references.pmid: 11341830
references.citation: J Biol Chem. 1951 Nov;193(1):265-75
references.pmid: 14907713
references.citation: Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1474-8
references.pmid: 8434007
references.citation: FEBS Lett. 1996 Jul 22;390(2):211-6
references.pmid: 8706862
references.citation: Eur J Biochem. 1994 Sep 1;224(2):533-40
references.pmid: 7925369
references.citation: J Mol Biol. 1993 Dec 5;234(3):779-815
references.pmid: 8254673