Fig. 4: Catalytic activity of the N domain and cyanophycin synthesis by dimeric CphA1.

a Mass spectra of (β-Asp-Arg)₈-NH₂, an 8mer cyanophycin segment in which the terminal carboxylate is replaced by an amide, before (left) and after (right) incubation with WT SuCphA1. After incubation with enzyme, the peak corresponding to (β-Asp-Arg)₈ (expected at 2187.0 Da) is reduced, and two peaks with sizes matching to (β-Asp-Arg)₄ (expected at 1102.5 Da) and (β-Asp-Arg)₄-NH₂ (expected at 1101.5 Da) appear. Peaks corresponding to Na⁺ and K⁺ adducts are also labeled. Peaks at 1093.4 and 1112.2 Da are deconvolution artifacts (Supplementary Fig. 5). b Mass spectra of (β-Asp-Arg)₈-Asn before (left) and after (right) incubation with WT SuCphA1. After incubation with enzyme, the peak corresponding to (β-Asp-Arg)₈-Asn (expected at 2301.1 Da) disappears and two peaks with sizes matching to (β-Asp-Arg)₄ (expected at 1102.5 Da) and (β-Asp-Arg)₄-Asn (expected at 1216.6 Da) appear. c, d Activity rate and lag time of SuCphA1 dimer complementation assays with c and without d primer. In presence of exogenous primer, the two mutant combinations G⁻M⁻N⁻/G⁺M⁺N⁺ and G⁻M⁺N⁺/G⁺M⁻N⁻ display a similar activity rate, although the G⁻M⁺N⁺/G⁺M⁻N⁻ combination displays a somewhat longer lag time. In the absence of primer, the G⁻M⁻N⁻/G⁺M⁺N⁺ combination displays a somewhat higher activity rate and lower lag time. n = 4 independent experiments. Data are presented as individual measurements and mean value, error bars represent SD values. e Activity assays and activity rate comparison of WT SuCphA1 (tetramer) and the W672A mutant (dimer) with (+) and without (−) primer. The two enzymes display similar primer-dependent activity, but the dimer has lower primer-independent activity. n = 4 independent experiments. Data are presented as individual measurements and mean value, error bars represent SD values.