A novel Dps‐type protein from insect gut bacteria catalyses hydrolysis and synthesis of N‐acyl amino acids

Abstract

A novel type of a microbial N-acyl amino acid hydrolase (AAH) from insect gut bacteria was purified, cloned and functionally characterized. The enzyme was obtained from Microbacterium arborescens SE14 isolated from the foregut of larvae of the generalist herbivore Spodoptera exigua. The substrates of AAH are N-acyl-glutamines previously reported to elicit plant defence reactions after introduction into the leaf during feeding. The isolated AAH catalyses the hydrolysis of the amide bond (K(m) = 36 micromol l(-1)) and, less efficient, the formation (K(m) = 3 mmol l(-1)) of the elicitor active N-acyl amino acids. The AAH from M. arborescens SE14 shows no homology to known fatty acyl amidases (EC 3.5.1.4) but belongs to the family of Dps proteins (DNA-binding protein from starved cell). In line with other DPS proteins AAH is a homododecamer (monomer 17 181 Da) and contains iron atoms (c. 1-16 iron atoms per subunit). Unlike genuine DPS proteins the enzyme does not significantly bind DNA. Amino acid hydrolase is the first member of the DPS family that catalyses the cleavage or formation of amide bonds. The participation of a microbial enzyme in the homeostasis of N-acyl-glutamines in the insect gut adds further complexity to the interaction between plants and their herbivores.

Structure and Mechanism of Iron Translocation by a Dps Protein from Microbacterium arborescens

“…Protein Purification-The purification of AAH was described in an earlier study (31). In brief, M. arborescens strain Se14 was grown in BHI medium for 18 h. Cells were harvested by centrifugation and disrupted by ultrasonication in 50 mM Tris-buffer (pH 8).…”

“…The final removal of small contaminants was performed by size exclusion chromatography over a Superdex 200 column (GE Healthcare). The activity of the samples was controlled during this procedure using the conjugation activity assay described in Ping et al (31). Only active fractions were used for further experiments.…”

“…Microorganisms living in the midgut have to compete with ROS and antimicrobial peptides (31). Some Dps proteins are known to act as peroxidases and detoxify radicals (6).…”

“…Some Dps proteins are known to act as peroxidases and detoxify radicals (6). M. arborescens uses the protein amino acid hydrolase (AAH), a Dps homolog, to both hydrolyze and synthesize N-acylamino acids (31). This class of amino acid conjugates with fatty acids plays an important role in the ability of plants to recognize herbivory (32) and induces the emission of volatile organic compounds (33).…”

“…This class of amino acid conjugates with fatty acids plays an important role in the ability of plants to recognize herbivory (32) and induces the emission of volatile organic compounds (33). Moreover, depending on the iron content of the medium, the Dps protein from M. arborescens displays different amounts of stored iron oxides as determined by inductively coupled plasma optical emission spectrometry (31).…”