A cryptic third active site in cyanophycin synthetase creates primers for polymerization

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine. Cyanophycin synthetase CphA1 polymerizes Asp and Arg into the nitrogen reserve polymer cyanophycin using two active sites. Sharon et al. show CphA1 has a cryptic 3rd active site that cleaves cyanophycin into primers for self-sufficient biosynthesis.

Automated summary

Cyanophycin synthetase CphA1 is capable of synthesizing cyanophycin without exogenous primers, utilizing a cryptic metallopeptidase-like active site in its N-terminal domain to digest cyanophycin into primers. Primer dependence of CphA1 in heterologous hosts may be a limiting factor for cyanophycin production.