title: S-adenosylmethionine-binding properties of a bacterial phospholipid N-methyltransferase.
abstract: The presence of the membrane lipid phosphatidylcholine (PC) in the bacterial membrane is critically important for many host-microbe interactions. The phospholipid N-methyltransferase PmtA from the plant pathogen Agrobacterium tumefaciens catalyzes the formation of PC by a three-step methylation of phosphatidylethanolamine via monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine. The methyl group is provided by S-adenosylmethionine (SAM), which is converted to S-adenosylhomocysteine (SAH) during transmethylation. Despite the biological importance of bacterial phospholipid N-methyltransferases, little is known about amino acids critical for binding to SAM or phospholipids and catalysis. Alanine substitutions in the predicted SAM-binding residues E58, G60, G62, and E84 in A. tumefaciens PmtA dramatically reduced SAM-binding and enzyme activity. Homology modeling of PmtA satisfactorily explained the mutational results. The enzyme is predicted to exhibit a consensus topology of the SAM-binding fold consistent with cofactor interaction as seen with most structurally characterized SAM-methyltransferases. Nuclear magnetic resonance (NMR) titration experiments and (14)C-SAM-binding studies revealed binding constants for SAM and SAH in the low micromolar range. Our study provides first insights into structural features and SAM binding of a bacterial phospholipid N-methyltransferase.
authors: Ruhr-Universität Bochum, Bochum, Germany.
authors: Meriyem
authors: M
authors: Aktas
authors: Jan
authors: J
authors: Gleichenhagen
authors: Raphael
authors: R
authors: Stoll
authors: Franz
authors: F
authors: Narberhaus
references: Mol Microbiol. 2006 Nov;62(3):906-15
references: 17010159
references: J Mol Biol. 2007 Oct 5;372(5):1227-45
references: 17716689
references: Protein Sci. 2003 Jul;12(7):1432-42
references: 12824489
references: J Biol Chem. 1979 May 25;254(10):3886-91
references: 438165
references: Mol Microbiol. 2001 Mar;39(5):1186-98
references: 11251836
references: Biomol NMR Assign. 2007 Jul;1(1):105-8
references: 19636840
references: Biopolymers. 2008 May;89(5):392-400
references: 17896349
references: Methods Enzymol. 1996;266:540-53
references: 8743705
references: J Mol Biol. 1986 May 5;189(1):113-30
references: 3537305
references: J Mol Biol. 1993 Jul 20;232(2):584-99
references: 8345525
references: BMC Struct Biol. 2008 May 13;8:25
references: 18477405
references: J Mol Biol. 1995 Nov 3;253(4):618-32
references: 7473738
references: Mol Cell. 2000 Aug;6(2):349-60
references: 10983982
references: Annu Rev Biophys Biomol Struct. 1995;24:293-318
references: 7663118
references: J Lipid Res. 2009 Apr;50 Suppl:S69-73
references: 18955729
references: Cell Microbiol. 2008 Feb;10(2):514-28
references: 17979985
references: Can J Biochem Physiol. 1959 Aug;37(8):911-7
references: 13671378
references: J Biol Chem. 1964 Jun;239:1705-13
references: 14217872
references: J Biol Chem. 1996 Mar 22;271(12):6794-800
references: 8636102
references: J Biomol NMR. 2000 May;17(1):87-8
references: 10909871
references: Nat Protoc. 2010 Apr;5(4):725-38
references: 20360767
references: BMC Struct Biol. 2005 Oct 14;5:19
references: 16225687
references: J Biotechnol. 2001 Oct 4;91(2-3):211-21
references: 11566392
references: J Bacteriol. 2009 Apr;191(7):2033-41
references: 19181804
references: Biochim Biophys Acta. 1994 Apr 14;1212(1):26-42
references: 8155724
references: Arch Biochem Biophys. 1994 May 1;310(2):417-27
references: 8179327
references: FEBS Lett. 2008 Sep 3;582(20):3005-10
references: 18692051
references: J Biol Chem. 1988 Nov 15;263(32):16864-71
references: 3182819
references: BMC Bioinformatics. 2008 Jan 23;9:40
references: 18215316
references: RNA. 2007 Nov;13(11):1969-76
references: 17872509
references: Curr Opin Struct Biol. 2002 Dec;12(6):783-93
references: 12504684
references: Prog Lipid Res. 2003 Mar;42(2):115-62
references: 12547654
references: J Biol Chem. 1995 Feb 17;270(7):2901-5
references: 7852367
references: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W500-2
references: 15215436
references: J Biol Chem. 2003 Sep 12;278(37):35826-36
references: 12842883
references: Biomol NMR Assign. 2007 Jul;1(1):45-7
references: 19636822
references: Nat Struct Biol. 2001 Mar;8(3):271-9
references: 11224575
references: J Biol Chem. 2004 Dec 10;279(50):52075-81
references: 15456775
references: J Bacteriol. 2009 Jan;191(1):365-74
references: 18978052
references: Biochim Biophys Acta. 1997 Sep 4;1348(1-2):142-50
references: 9370326
references: J Mol Biol. 1995 Mar 17;247(1):16-20
references: 7897657
references: Biochim Biophys Acta. 1996 Nov 12;1277(1-2):115-126
references: 30897681
references: J Bacteriol. 2008 Sep;190(17):5855-61
references: 18586937
references: Anal Biochem. 1996 Mar 1;235(1):1-10
references: 8850540
references: J Mol Biol. 1983 Jun 5;166(4):557-80
references: 6345791
references: J Biol Chem. 2002 Nov 8;277(45):42549-56
references: 12196515
references: Eur J Cell Biol. 2010 Dec;89(12):888-94
references: 20656373
references: Cell. 1993 Jul 30;74(2):299-307
references: 8343957
references: Proteins. 2009;77 Suppl 9:100-13
references: 19768687
references: J Bacteriol. 2008 Jan;190(2):571-80
references: 17993534
references: J Biol Chem. 1993 Aug 5;268(22):16655-63
references: 8344945
references: Cell Microbiol. 2006 Aug;8(8):1322-35
references: 16882035