An interdomain disulfide bridge links the NtA and first FS domain in agrin

Agrin is a multidomain heparan sulfate proteoglycan involved in postsynaptic differentiation at the neuromuscular junction. Binding of agrin to synaptic basal lamina is mediated by the N-terminal agrin (NtA) domain. The NtA domain of agrin is followed by a tandem of nine follistatin-like (FS) domains forming a rod-like spacer to the laminin G-like domains of the molecule. Here we report that the most C-terminal cysteine residue of NtA (Cys123) forms an interdomain disulfide bond with the FOLN subdomain of the FS module. Remarkably, this single cysteine is flanked by Leu117 and Val124, which are two essential β-branched amino acids forming the heterocomplex of NtA with the γ1 chain of laminin. Moreover, we show that this covalent linkage compensates for the seven amino acid residue splice insert at the very C-terminal helix H3 and causes a rigid interface between NtA and FS independent of the alternative mRNA splice event. These results suggest that the interdomain disulfide bond between the NtA and the first FS domain might be important for the proper folding of agrin

Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate beta-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg(386) interacts with the alpha-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydrogen bond with O-3' of the cofactor; and Arg(292) interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k(cat)' for beta-decarboxylation of L-aspartate was 0.08 s(-1), whereas k(cat)' for transamination was decreased to 0.01 s(-1). AspAT was thus converted into an L-aspartate beta-decarboxylase that catalyzes transamination as a side reaction. The major pathway of beta-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low beta-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits beta-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5'-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions