Resistance to the antibiotic thiostrepton, in producing Streptomycetes, is conferred by the S-adenosyl-l-methionine (SAM)-dependent SPOUT methyltransferase Tsr. For this and related enzymes, the roles of active site amino acids have been inadequately described. Herein, we have probed SAM interactions in the Tsr active site by investigating the catalytic activity and the thermodynamics of SAM binding by site-directed Tsr mutants. Two arginine residues were demonstrated to be critical for binding, one of which appears to participate in the catalytic reaction. Additionally, evidence consistent with the involvement of an asparagine in the structural organization of the SAM binding site is presented.

Background and aims: Factor (F)XIII is a protransglutaminase that on activation by thrombin introduces covalent cross-links in fibrin and incorporates inhibitors of fibrinolysis to increase resistance of the clot to proteolysis. Activation of FXIII involves cleavage of R37-G38 in the A-subunit by thrombin. Interaction with thrombin has been investigated for peptides based on FXIII-A sequence, but not for full-length FXIII-A. The only information on full-length FXIII is derived from the naturally occurring V34L variant, which has been shown to increase activation rates. We therefore used site-directed mutagenesis on recombinant FXIII-A to investigate thrombin interaction. Methods: Recombinant full-length FXIII-A was expressed with an N-terminal GST-tag in E coli. The cells expressed fully functional FXIII-A of dimeric form. Site-directed mutagenesis was used to produce mutants of the activation peptide and the region downstream of the thrombin cleavage site. Residues 28-35 and 37-42 were mutated to alanine, V29 also to leucine, and V34 to leucine and methionine, to investigate the role of increase of side-chain mass at these positions. Recombinant proteins were purified to homogeneity using glutathione affinity chromatography. Interaction with thrombin was investigated through generation of cross-linking activity in a biotin-pentylamine incorporation assay. Cleavage of the recombinant proteins by thrombin was confirmed by SDS-PAGE. Results: Mutation of R37 at the thrombin cleavage site to alanine completely abolished activation of FXIII-A by thrombin as expected. Mutation of residues V29, E30, G33 and V34 to alanine significantly reduced activation rates of FXIII-A, indicating a role for these residues of the activation peptide just upstream of the thrombin cleavage site in the interaction between FXIII-A and thrombin. There were no significant effect of mutations of residues G38, V39, N40, L41 or Q42 to alanine on activation rates of FXIII-A, showing that residues immediately downstream of the thrombin cleavage site do not play a role. V34L increased activation rate by thrombin. A similar effect was observed for V29L. Mutation of V34 to different residues showed that an increase in side-chain mass increased activation rates of FXIII-A by thrombin (M>L>V>A34). Conclusion: These data demonstrate that residues R37, V34 and G33 at P1, P4 and P5 positions play a role in full-length FXIII-A for thrombin interaction. They also indicate a further role for residues V29 and E30 in this process. Side-chain mass at residue 34 enhances thrombin interaction, elucidating a mechanism by which V34L increases FXIII activation rates.