Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro
Tóm tắt
Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD+ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD+, especially for one of the alleles, nadD72. In this study these two mutant proteins have been further characterized together with ten new mutant variants. Of the, in total, twelve mutations four are in a conserved motif in the C-terminus and eight are in the active site. We have tested the activity of the enzymes in vitro and their effect on the growth phenotype in vivo. There is a very good correlation between the two data sets. The mutations in the C-terminus did not reveal any function for the conserved motif. On the other hand, our data has lead us to assign amino acid residues His-19, Arg-46 and Asp-109 to the active site. We have also shown that the nadD gene is essential for growth in E. coli.
Tài liệu tham khảo
Berger F, Ramirez-Hernandez MH, Ziegler M: The life of a centenarian: signalling functions of NAD(P). Trends Biochem Sci. 2004, 29: 111-118. 10.1016/j.tibs.2004.01.007.
Rizzi M, Schindelin H: Structural biology of enzymes involved in NAD and molybdenum cofactor synthesis. Curr Opin Struct Biol. 2002, 12: 709-720. 10.1016/S0959-440X(02)00385-8.
Hilz H: ADP-ribose. A historical overview. Adv Exp Med Biol. 1997, 419: 15-24.
Lee HC: NAADP: An emerging calcium signaling molecule. J Membr Biol. 2000, 173: 1-8. 10.1007/s002320001001.
Singleton MR, Hakansson K, Timson DJ, Wigley DB: Structure of the adenylation domain of an NAD+-dependent DNA ligase. Structure Fold Des. 1999, 7: 35-42. 10.1016/S0969-2126(99)80007-0.
Magni G, Amici A, Emanuelli M, Raffaelli N: Enzymology of NAD+ synthesis. Advances in Enzymology and Related Areas of Molecular Biology. Edited by: Purich DL. 1999, , J Wiley & Sons, Inc., 73: 135-182.
Begley TP, Kinsland C, Mehl RA, Osterman A, Dorrestein P: The biosynthesis of nicotinamide adenine dinucleotides in bacteria. Vitam Horm. 2001, 61: 103-119.
Gerdes SY, Scholle MD, D'Souza M, Bernal A, Baev MV, Farrell M, Kurnasov OV, Daugherty MD, Mseeh F, Polanuyer BM, Campbell JW, Anantha S, Shatalin KY, Chowdhury SA, Fonstein MY, Osterman AL: From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways. J Bacteriol. 2002, 184: 4555-4572. 10.1128/JB.184.16.4555-4572.2002.
Mehl RA, Kinsland C, Begley TP: Identification of the Escherichia coli nicotinic acid mononucleotide adenylyltransferase gene. J Bact. 2000, 182: 4372-4374. 10.1128/JB.182.15.4372-4374.2000.
Zhang H, Zhou T, Kurnasov O, Cheek S, Grishin NV, Osterman A: Crystal Structures of E. coli Nicotinate Mononucleotide Adenylyltransferase and Its Complex with Deamido-NAD. Structure. 2002, 10: 69-79. 10.1016/S0969-2126(01)00693-1.
Bork P, Holm L, Koonin EV, Sander C: The cytidylyltransferase superfamily: identification of the nucleotide-binding site and fold prediction. Proteins. 1995, 22: 259-266. 10.1002/prot.340220306.
D'Angelo I, Raffaelli N, Dabusti V, Lorenzi T, Magni G, Rizzi M: Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD+ biosynthesis. Structure. 2000, 8: 993-1004. 10.1016/S0969-2126(00)00190-8.
Saridakis V, Christendat D, Kimber MS, Dharamsi A, Edwards AM, Pai EF: Insights into ligand binding and catalysis of a central step in NAD+ synthesis: structures of Methanobacterium thermoautotrophicum NMN adenylyltransferase complexes. J Biol Chem. 2001, 276: 7225-7232. 10.1074/jbc.M008810200.
Saridakis V, Pai EF: Mutational, structural, and kinetic studies of the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase. J Biol Chem. 2003, 278: 34356-34363. 10.1074/jbc.M205369200.
Stancek M, Isaksson LA, Ryden-Aulin M: fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli. Microbiology. 2003, 149: 2427-2433. 10.1099/mic.0.26337-0.
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL: An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A. 2000, 97: 5978-5983. 10.1073/pnas.100127597.
Hughes KT, Ladika D, Roth JR, Olivera BM: An indispensable gene for NAD biosynthesis in Salmonella typhimurium. J Bact. 1983, 155: 213-221.
Zhou T, Kurnasov O, Tomchick DR, Binns DD, Grishin NV, Marquez VE, Osterman A, Zhang H: Structure of Human Nicotinamide/Nicotinic Acid Mononucleotide Adenylyltransferase. J Biol Chem. 2002, 277: 13148-13154. 10.1074/jbc.M111469200.
Garavaglia S, D'Angelo I, Emanuelli M, Carnevali F, Pierella F, Magni G, Rizzi M: Structure of human NMN adenylyltransferase. A key nuclear enzyme for NAD homeostasis. J Biol Chem. 2002, 277: 8524-8530. 10.1074/jbc.M111589200.
Park YS, Gee P, Sanker S, Schurter EJ, Zuiderweg ER, Kent C: Identification of functional conserved residues of CTP:glycerol-3-phosphate cytidylyltransferase. Role of histidines in the conserved HXGH in catalysis. J Biol Chem. 1997, 272: 15161-15166. 10.1074/jbc.272.24.15161.
Veitch DP, Gilham D, Cornell RB: The role of histidine residues in the HXGH site of CTP:phosphocholine cytidylyltransferase in CTP binding and catalysis. Eur J Biochem. 1998, 255: 227-234. 10.1046/j.1432-1327.1998.2550227.x.
Miller JH: Formulas and recipes. Experiments in Molecular Genetics. 1972, Cold Spring Harbor, Cold Spring Harbor Laboratory, 433-
Sambrook J, Fritsch EF, Maniatis T: A Laboratory Manual. 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 2nd
Nilsson B, Moks T, Jansson B, Abrahmsen L, Elmblad A, Holmgren E, Henrichson C, Jones TA, Uhlen M: A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng. 1987, 1: 107-113.
Woodcock DM, Crowther PJ, Doherty J, Jefferson S, DeCruz E, Noyer-Weidner M, Smith SS, Michael MZ, Graham MW: Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 1989, 17: 3469-3478.
Peptide properties calculator. [http://www.basic.nwer.edu/biotools/proteincalc.html]
Delano WL: The PyMOL Molecular Graphics System (2002) on World Wide Web. [http://http:llwww.pymol.org]