Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis.
5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is important in a number of cellular functions such as polyamine biosynthesis, methionine salvaging, biological methylation, and quorum sensing. The nucleosidase is found in many microbes but not in mammalian systems, thus making MTAN a broad-spectrum antimicrobial drug target. Substrate binding and catalytic residues were identified from the crystal structure of MTAN complexed with 5'-methylthiotubercidin [Lee, J. E., Cornell, K. A., Riscoe, M. K. and Howell, P. L. (2003) J. Biol. Chem. 278 (10) 8761-8770]. The roles of active site residues Met9, Glu12, Ile50, Ser76, Val102, Phe105, Tyr107, Phe151, Met173, Glu174, Arg193, Ser196, Asp197, and Phe207 have been investigated by site-directed mutagenesis and steady-state kinetics. Mutagenesis of residues Glu12, Glu174, and Asp197 completely abolished activity. The location of Asp197 and Glu12 in the active site is consistent with their having a direct role in enzyme catalysis. Glu174 is suggested to be involved in catalysis by stabilizing the transition state positive charge at the O3', C2', and C3' atoms and by polarizing the 3'-hydroxyl to aid in the flow of electrons to the electron withdrawing purine base. This represents the first indication of the importance of the 3'-hydroxyl in the stabilization of the transition state. Furthermore, mutation of Arg193 to alanine shows that the nucleophilic water is able to direct its attack without assistance from the enzyme. This mutagenesis study has allowed a reevaluation of the catalytic mechanism.
Lee
Jeffrey E
JE
Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Luong
Winnie
W
Huang
David J T
DJ
Cornell
Kenneth A
KA
Riscoe
Michael K
MK
Howell
P Lynne
PL
D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D015317:Biogenic Polyamines / Q000096:biosynthesis; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006714:Homoserine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D007700:Kinetics; D007783:Lactones; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013872:Thionucleosides / Q000737:chemistry / Q000378:metabolism; D014372:Tubercidin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D015317:Biogenic Polyamines; D029968:Escherichia coli Proteins; D007783:Lactones; C091088:N-octanoylhomoserine lactone; D013872:Thionucleosides; C021716:5'-methylthiotubercidin; D006714:Homoserine; D008715:Methionine; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase; D014372:Tubercidin