Biosynthesis of the Stress-Protectant and Chemical Chaperon Ectoine: Biochemistry of the Transaminase EctB

Stress Protectant Secondary Metabolites and their Metabolic Engineering to Enhance Abiotic Stress Tolerance in Plants

In vitro Plant Breeding towards Novel Agronomic Traits ◽

10.1007/978-981-32-9824-8_11 ◽

2019 ◽

pp. 197-216

Author(s):

Gurminder Kaur ◽

Deepak Ganjewala

Keyword(s):

Abiotic Stress ◽

Metabolic Engineering ◽

Secondary Metabolites ◽

Stress Tolerance ◽

Abiotic Stress Tolerance ◽

Stress Protectant

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The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011277 ◽

2020 ◽

Vol 295 (9) ◽

pp. 2822-2838 ◽

Cited By ~ 3

Author(s):

Alexandra A. Richter ◽

Stefanie Kobus ◽

Laura Czech ◽

Astrid Hoeppner ◽

Jan Zarzycki ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Catalytic Core ◽

Chemical Chaperone ◽

Practical Applications ◽

Acetyl Group ◽

Function Relationship ◽

Thermotolerant Bacterium ◽

Stress Protectant ◽

Relationship Of

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2–2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

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Regulation of trehalose, a typical stress protectant, on central metabolisms, cell growth and division of Saccharomyces cerevisiae CEN.PK113-7D

Food Microbiology ◽

10.1016/j.fm.2020.103459 ◽

2020 ◽

Vol 89 ◽

pp. 103459

Author(s):

Xiaoru Zhang ◽

Yaxian Zhang ◽

Hao Li

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Stress Protectant

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Trehalose in yeast, stress protectant rather than reserve carbohydrate

Antonie van Leeuwenhoek ◽

10.1007/bf00548935 ◽

1990 ◽

Vol 58 (3) ◽

pp. 209-217 ◽

Cited By ~ 332

Author(s):

Andres Wiemken

Keyword(s):

Reserve Carbohydrate ◽

Stress Protectant

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Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of Cryptococcus neoformans

Infection and Immunity ◽

10.1128/iai.00624-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5877-5887 ◽

Cited By ~ 106

Author(s):

Elizabeth Wills Petzold ◽

Uwe Himmelreich ◽

Eleftherios Mylonakis ◽

Thomas Rude ◽

Dena Toffaletti ◽

...

Keyword(s):

High Temperature ◽

Cryptococcus Neoformans ◽

Pathogenic Fungi ◽

Growth Defect ◽

C Elegans ◽

As Stress ◽

Stress Protectant ◽

Survival Mechanisms ◽

Trehalose Synthesis

ABSTRACT The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37°C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37°C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

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Disruption of the Candida albicans TPS2 Gene Encoding Trehalose-6-Phosphate Phosphatase Decreases Infectivity without Affecting Hypha Formation

Infection and Immunity ◽

10.1128/iai.70.4.1772-1782.2002 ◽

2002 ◽

Vol 70 (4) ◽

pp. 1772-1782 ◽

Cited By ~ 82

Author(s):

Patrick Van Dijck ◽

Larissa De Rop ◽

Karolina Szlufcik ◽

Elke Van Ael ◽

Johan M. Thevelein

Keyword(s):

Candida Albicans ◽

High Throughput Screening ◽

Deletion Mutant ◽

Systemic Infection ◽

Homozygous Deletion ◽

Antifungal Drugs ◽

Gene Encoding ◽

Complete Inactivation ◽

Hypha Formation ◽

Stress Protectant

ABSTRACT Deletion of trehalose-6-phosphate phosphatase, encoded by TPS2, in Saccharomyces cerevisiae results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated whether Tre6P phosphatase could be a useful target for antifungals in Candida albicans. We have cloned the C. albicans TPS2 (CaTPS2) gene and constructed heterozygous and homozygous deletion strains. As in S. cerevisiae, complete inactivation of Tre6P phosphatase in C. albicans results in 50-fold hyperaccumulation of Tre6P, thermosensitivity, and rapid death of the cells after a few hours at 44°C. As opposed to inactivation of Tre6P synthase by deletion of CaTPS1, deletion of CaTPS2 does not affect hypha formation on a solid glucose-containing medium. In spite of this, virulence of the homozygous deletion mutant is strongly reduced in a mouse model of systemic infection. The pathogenicity of the heterozygous deletion mutant is similar to that of the wild-type strain. CaTPS2 is a new example of a gene not required for growth under standard conditions but required for pathogenicity in a host. Our results suggest that Tre6P phosphatase may serve as a potential target for antifungal drugs. Neither Tre6P phosphatase nor its substrate is present in mammals, and assay of the enzymes is simple and easily automated for high-throughput screening.

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