Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase

Abstract

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.

Heterologous production of cyanophycin withTatumella morbiroseicyanophycin synthetase

“…PCC6308 ( 6308 CphA1) is well-characterized and often serves as a benchmark for heterologous cyanophycin production [18–21], yet the 6308 CphA1 enzyme itself does not robustly express and purify from E. coli . Recently, other CphA1 enzymes that do express well in E. coli and purify as soluble and stable samples were identified and used for structure determination and functional analysis by two authors of this work [3] and by others [22]. We hypothesized that CphA1 enzymes with robust and stable heterologous expression in E. coli would in turn support enhanced cyanophycin production within that host.…”

Structure and function of a hexameric cyanophycin synthetase 2

“…We also report the first substrate co-complex structures of a CphA2 enzyme. That binding of cyanophycin to the CphA2 G domain is very similar to its binding to the CphA1 G domain (Miyakawa et al, 2022;Sharon et al, 2021) is not surprising, since CphA2 evolved from CphA1, and the G domain retains very similar activity. Cyanophycin binding is dominated by electrostatic interactions for both enzymes, which feature similar, shallow active sites (Supporting Information Figure S6B), rather than the deep substrate binding pockets seen in some other enzymes.…”

“…Cyanophycin binding is dominated by electrostatic interactions for both enzymes, which feature similar, shallow active sites (Supporting Information Figure S6B), rather than the deep substrate binding pockets seen in some other enzymes. Our inability to capture incoming β‐Asp‐Arg substrate precludes a comparison of its binding with that of Asp (Miyakawa et al, 2022; Sharon, Pinus, et al, 2022). It is likely a subtle, cryptic change in the G domain large loop that provides specificity for β‐Asp‐Arg over Asp.…”

Structure and function of a hexameric cyanophycin synthetase 2

“…We also report the first substrate co-complex structures of a CphA2 enzyme. That binding of cyanophycin to the CphA2 G domain is very similar to its binding to the CphA1 G domain (Sharon et al 2021;Miyakawa et al 2022) is not surprising, since CphA2 evolved from CphA1, and the G domain retains very similar activity. Cyanophycin binding is dominated by electrostatic interactions for both enzymes, which feature similar, shallow active sites (Supplemental Figure 2C), rather than the deep substrate binding pockets seen in some other enzymes.…”