Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Mar 13;114(2):27.
doi: 10.1007/s11103-024-01416-1.

Arginine inhibits the arginine biosynthesis rate-limiting enzyme and leads to the accumulation of intracellular aspartate in Synechocystis sp. PCC 6803

Noriaki Katayama  1 , Takashi Osanai  2
Affiliations

Arginine inhibits the arginine biosynthesis rate-limiting enzyme and leads to the accumulation of intracellular aspartate in Synechocystis sp. PCC 6803

Noriaki Katayama et al. Plant Mol Biol. .

Abstract
in English, Spanish

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.

This study revealed the catalytic efficiency and inhibition of cyanobacterial argininosuccinate synthetase by arginine and demonstrated that a strain overexpressing this enzyme grew faster than the wild-type strain.

Keywords: Synechocystis; Arginine biosynthesis; Argininosuccinate synthetase; Cyanobacteria.

PubMed Disclaimer

Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose.

Figures

Fig. 1
Fig. 1
Summary of arginine biosynthesis pathway around reactions catalyzed by ArgG in Synechocystis 6803. This reaction in arginine biosynthesis is based on the previous study (Flores et al. 2019). The gray ellipse represented arginine biosynthesis enzymes. The orange ellipse represented ArgG in this study detailed biochemical analysis was performed. Glu, glutamate; N-AcGlu, N-acetyl-glutamate; N-AcGluP, N-acetyl-glutamyl phosphate; N-AcGluSS, N-acetyl-glutamate semialdehyde; N-AcOrn, N-acetyl-ornithine; Orn, ornithine; Carbamoyl-P, carbamoyl phosphate; Cit, citrulline; Asp, aspartate; AS, argininosuccinate; Arg, arginine; Fum, fumarate
Fig. 2
Fig. 2
Construction of the ArgGOX and ArgHOX strains. (a) Vectors for establishing the ArgGOX and ArgHOX strains. (b) The expression levels of SyArgG and SyArgH in the GT, ArgGOX, and ArgHOX strains. The data represent the relative amounts of transcript products, and the amount in the GT strain was set at 100%. Data represent the means ± SD obtained from four or five independent experiments. (c) The expression levels of arginine biosynthesis-related genes in the GT, ArgGOX, and ArgHOX strains. The data represent the relative amounts of transcript products, and the amount in the GT strain was set at 100%. Data represent the means ± SD obtained from six independent experiments. Statistically significant differences between the activity in the absence or presence of the effector were examined by two-tailed Student’s t-test and are represented by asterisks (* = P < 0.05, ** = P < 0.005)
Fig. 3
Fig. 3
Purification of SyArgG and determination of its optimal temperature and pH. (a) Purified GST-tagged SyArgG separated on 12% SDS-PAGE gel and stained with Quick Staining Solution. (b) Effect of temperature on SyArgG activity. (c) Effect of pH on SyArgG activity in various buffers. Blue circles, orange squares, gray triangles, and yellow rhombuses represent MES-NaOH, Tris-HCl, CHES-KOH, and CAPS-KOH, respectively
Fig. 4
Fig. 4
Determination of Km values for aspartate, citrulline, and ATP. and the effect of various metabolites on SyArgG activity. This measurement was performed at 38 °C and pH 9.0. SyArgG activity was measured by varying (a) sodium aspartate, (b) citrulline, and (c) ATP concentrations. One unit of SyArgG activity was defined as a consumption of 1 μmol NADH per minute. Data represent the means ± SD obtained from the triplicated independent experiments
Fig. 5
Fig. 5
Effect of various metabolites on SyArgG activity. This measurement was performed at 38 °C and pH 9.0 and fixed concentrations of aspartate, citrulline, and ATP were 7.5, 7.5, and 1.0 mM respectively. SyArgG activity was represented by relative activity, whereby the activity in the absence of metabolites was set at 100%. Data represent the means ± SD obtained from triplicate independent experiments. Statistically significant differences between the activity in the absence or presence of the effector were examined by two-tailed Student’s t-test and are represented by asterisks (* = P < 0.05, ** = P < 0.005, *** = P < 0.0005). Glu, glutamate; Gln, glutamine; Asn, asparagine; Orn, ornithine; Arg, arginine; Lys, lysin; Pro, proline; 2-OG, 2-oxoglutarate; Cit, citrate; Mal, malate; Fum, fumarate; Suc, succinate; Isoc, isocitrate
Fig. 6
Fig. 6
Growth curves of the GT, ArgGOX, and ArgHOX strains under photoautotrophic conditions with (a) NaNO3 and (b) arginine as nitrogen source and intracellular aspartate concentration. The blue circle, orange square, and gray triangle represent the GT, ArgGOX, and ArgHOX strains, respectively. (c) Intracellular aspartate concentration in the GT, ArgGOX, and ArgHOX strains with different nitrogen sources. Data represent the means ± SD obtained from triplicate independent experiments. Statistically significant differences between the OD730 in the GT, ArgGOX, or ArgHOX strains were examined by paired two-tailed Student’s t-tests and are represent by asterisks (* = P < 0.05, ** = P < 0.005)
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Bolay P, Rozbeh R, Muro-Pastor MI, Timm S, Hagemann M, Florencio FJ, Forchhammer K, Klähn S. The novel PII-interacting protein PirA controls flux into the cyanobacterial ornithine-ammonia cycle. mBio. 2021;12(2):e00229–e00221. doi: 10.1128/mbio.00229-21. - DOI - PMC - PubMed
    1. Burgstaller H, Wang Y, Caliebe J, Hueren V, Appel J, Boehm M, Leitzke S, Theune M, King PW, Gutekunst K. Synechocystis sp. PCC 6803 requires bidirectional hydrogenase to metabolize glucose and arginine under oxic conditions. Front Microbiol. 2022;13:896190. doi: 10.3389/fmicb.2022.896190. - DOI - PMC - PubMed
    1. Esteves-Ferreira AA, Cavalcanti JHF, Vaz MGMV, Alvarenga LV, Nunes-Nesi A, Araújo WL. Cyanobacterial nitrogenases: phylogenetic diversity, regulation, and functional predictions. Genet Mol Biol. 2017;40:261–275. doi: 10.1590/1678-4685-GMB-2016-0050. - DOI - PMC - PubMed
    1. Esteves-Ferreira AA, Inaba M, Fort A, Araújo WL, Sulpice R. Nitrogen metabolism in cyanobacteria: metabolic and molecular control, growth consequences and biotechnological applications. Crit Rev Microbiol. 2018;44:541–560. doi: 10.1080/1040841X.2018.1446902. - DOI - PubMed
    1. Flombaum P, Gallegos JL, Gordillo RA, Rincón J, Zabala LL, Jiao N, Karl DM, Li WK, Lomas MW, Veneziano D, Vera CS, Vrugt JA, Martiny AC. Present and future global distributions of the marine cyanobacteria Prochlorococcus and Synechococcus. Proc Natl Acad Sci USA. 2013;110:9824–9829. doi: 10.1073/pnas.1307701110. - DOI - PMC - PubMed

LinkOut - more resources

-