Department of Biochemistry and Centre de recherche en biologie structurale, McGill University, Montréal, QC, H3G 0B1, Canada.
Department of Chemistry, McGill University, Montreal, QC, H3A 0B8, Canada.
Nat Commun. 2022 Jul 7;13(1):3923. doi: 10.1038/s41467-022-31542-7.
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.
蓝藻素是许多细菌中一种含氮的储备生物聚合物，具有广阔的工业应用前景。它由蓝藻素合成酶 1（CphA1）合成，具有聚-L-天冬氨酸主链，每个天冬氨酸侧链上连接一个 L-精氨酸残基。人们认为，CphA1 通常需要蓝藻素的现有片段作为蓝藻素聚合的引物。在这项研究中，我们表明，大多数 CphA1 不需要外源引物，并发现了引物独立性的惊人原因：CphA1 可以在没有引物的情况下产生少量的蓝藻素，而许多 CphA1 的 N 端结构域中存在一个意想不到的、隐蔽的金属肽酶样活性位点，可将这些蓝藻素消化成引物，从而解决了引物可用性的问题。我们提出了共复合物冷冻电镜结构，进行了突变，使 CphA1 在引物依赖性和非依赖性之间转换，并证明在异源宿主中，引物依赖性可能是蓝藻素生产的限制因素。在 CphA1 中，具有相反催化活性的结构域结合在一起，形成了一个非凡的、自给自足的生物合成纳米机器。
