Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin

Abstract

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into β-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade β-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and β-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its β-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the “arginine/ornithine transport” (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa . In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use β-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Cyanophycin and its biosynthesis: not hot but very cool

“…We recently discovered that the human pathogen Pseudomonas aeruginosa and many other Pseudomonas species encode the ability to import and survive on cyanophycin-derived material. 6 Their aot operon encodes a multi-subunit arginine transporter (AotJQMP), an arginine-dependent transcription activator (ArgR), and a previously uncharacterized enzyme, AotO. 83,84 We found AotO to be a member of a new family of cyanophycin dipeptide hydrolase enzymes (see Section 4.4.1) specific for β-Asp-Arg/Lys, and that AotJQMP can transport β-Asp-Arg in addition to Arg.…”

“…5 Outlookthe final frontiers for cyanophycin research Cyanophycin metabolism genes are much more common than might be expected: Around 11% of complete bacterial genomes in the NCBI RefSeq database encode at least one gene for cyanophycin metabolism. 6 For comparison, ∼38% of these genomes encode at least one gene for glycogen metabolism. These numbers highlight how common it is for bacteria to be cyanophycin producers or scavengers, much more so than currently recognized.…”

“…First observed in 1878 as granules within cyanobacterial cells, 2,3 cyanophycin is produced by a wide range of bacteria and can be degraded by many more. [4][5][6] The nitrogen content of cyanophycin, 24% by mass, is higher than that of other biopolymers. 7 It is insoluble at physiological pH, and so spontaneously forms large, inert granules (Fig.…”

Structure and function of a hexameric cyanophycin synthetase 2

“…To use the nitrogen, carbon and energy stored in the polymer, cyanophycin must be degraded into its constituent amino acids. This process takes place in two steps: first, a specialized enzyme related to proteases, cyanophycinase, hydrolyses it into β-Asp-Arg dipeptides (Richter et al 1999); then, these dipeptides are hydrolyzed by one of several enzymes with isoaspartyl dipeptidase activity into free aspartate and arginine (Figure 1B) (Sharon et al 2023b;Sharon and Schmeing 2023).…”

Structure and function of a hexameric cyanophycin synthetase 2

“…To use the nitrogen, carbon, and energy stored in the polymer, cyanophycin must be degraded into its constituent amino acids. This process takes place in two steps: First, a specialized enzyme related to serine proteases, cyanophycinase, hydrolyses it into β‐Asp‐Arg dipeptides (Richter et al, 1999); then, these dipeptides are lysed into free aspartate and arginine by one of several enzymes with isoaspartyl dipeptidase activity (Figure 1b) (Sharon, McKay, et al, 2023; Sharon & Schmeing, 2023).…”