The Effects of Salts and Osmoprotectants on Enzyme Activities of Fructose-1,6-biphosphate Aldolases in a Halotolerant Cyanobacterium, Halothece sp. PCC 7418.

Siripat Ngoennet,Tanutcha Patipong,Hakuto Kageyama,Masaki Honda,Takashi Hibino,Rungaroon Waditee-Sirisattha

doi:10.3390/life10030023

Siripat Ngoennet, Tanutcha Patipong + Show 4 more

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https://doi.org/10.3390/life10030023

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Abstract

The halotolerant cyanobacterium, Halothece sp. PCC 7418, possesses two classes of fructose-1,6-bisphosphate aldolase (FBA): H2846 and H2847. Though class I (CI)-FBA H2846 is thought to be associated with salt tolerance, the regulatory mechanisms, molecular characteristics, and expression profiles between H2846 and class II (CII)-FBA H2847 have scarcely been investigated. Here, we show that the accumulation of the H2846 protein is highly responsive to both up- and down-shock with NaCl, whereas H2847 is constitutively expressed. The activity of CI- and CII-FBA in cyanobacterial extracts is correlated with the accumulation patterns of H2846 and H2847, respectively. In addition, it was found that these activities were inhibited by NaCl and KCl, with CII-FBA activity strikingly inhibited. It was also found that the CI-FBA activity of recombinant H2846 was hindered by salts and that this hindrance could be moderated by the addition of glycine betaine (GB), whereas no moderation occurred with other potential osmoprotectant molecules (proline, sucrose, and glycerol). In addition, a phylogenetic analysis showed that CI-FBAs with higher similarities to H2846 tended to be distributed among potential GB-synthesizing cyanobacteria. Taken together, our results provide insights into the independent evolution of the CI- and CII-FBA gene families, which show distinct expression profiles and functions following salt stress.

Highlights

IntroductionFructose-1,6-bisphosphate aldolase (FBA), an enzyme, which is indispensable for glycolysis, gluconeogenesis, and the Calvin cycle, catalyzes the reversible aldol cleavage of fructose
Fructose-1,6-bisphosphate aldolase (FBA), an enzyme, which is indispensable for glycolysis, gluconeogenesis, and the Calvin cycle, catalyzes the reversible aldol cleavage of fructose1,6-bisphosphate (FBP) into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) (Figure 1) [1]
To explore the relationship between these clades and their ability to synthesize glycine betaine (GB), we investigated the distribution of the GB-synthetic enzymes, glycine/sarcosine N-methyltransferase (GSMT) and dimethylglycine N-methyltransferase (DMT), among the cyanobacteria we analyzed

Summary

Introduction

Fructose-1,6-bisphosphate aldolase (FBA), an enzyme, which is indispensable for glycolysis, gluconeogenesis, and the Calvin cycle, catalyzes the reversible aldol cleavage of fructose. 1,6-bisphosphate (FBP) into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) (Figure 1) [1]. FBAs are classified into two distinct classes, according to their catalytic mechanism and independent evolutionary occurrence [2]. Class I (CI)-FBAs catalyze the reaction via the formation of a Schiff base, which attacks the C1 carbonyl group of G3P between the C2 carbonyl group of DHAP and the N6 amino group of an essential lysine residue in the active site. Class II (CII)-FBAs require divalent metal ions for a similar catalytic mechanism. The purification process consisted of two chromatographic steps: affinity purification that used an Ni‐NTA‐spin kit column (Qiagen, Hilden, Germany) and size‐exclusion chromatographic purification that used a HiLoad 16/600 Superdex 200

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