Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases.
Archaea contain a class of methionine adenosyltransferases (MATs) that exhibit substantially higher stability than their mesophilic counterparts. Their sequences are highly divergent, but preserve the essential active site motifs of the family. We have investigated the origin of this increased stability using chemical denaturation experiments on Methanococcus jannaschii MAT (Mj-MAT) and mutants containing single tryptophans in place of tyrosine residues. The results from fluorescence, circular dichroism, hydrodynamic, and enzyme activity measurements showed that the higher stability of Mj-MAT derives largely from a tighter association of its subunits in the dimer. Local fluorescence changes, interpreted using secondary structure predictions, further identify the least stable structural elements as the C-terminal ends of beta-strands E2 and E6, and the N-terminus of E3. Dimer dissociation however requires a wider perturbation of the molecule. Additional analysis was initially hindered by the lack of crystal structures for archaeal MATs, a limitation that we overcame by construction of a 3D-homology model of Mj-MAT. This model predicts preservation of the chain topology and three-domain organization typical of this family, locates the least stable structural elements at the flat contact surface between monomers, and shows that alterations in all three domains are required for dimer dissociation.
Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029 Madrid, Spain.
Francisco
F
Garrido
Carlos
C
Alfonso
John C
JC
Taylor
George D
GD
Markham
María A
MA
Pajares
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017931:DNA Primers; D004795:Enzyme Stability; D017017:Methanococcus / Q000201:enzymology*; D008716:Methionine Adenosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D008716:Methionine Adenosyltransferase