Functional Profiling and Crystal Structures of Isothiocyanate Hydrolases Found in Gut-Associated and Plant-Pathogenic Bacteria

Abstract

Isothiocyanates (ITCs) are produced by cruciferous plants to protect them against herbivores and infection by microbes. These compounds are of particular interest due to their antimicrobial and anticarcinogenic properties. The breakdown of ITCs in nature is catalyzed by isothiocyanate hydrolase (ITCase), a novel family within the metallo-β-lactamase-fold superfamily of proteins. genes that code for ITCases are particularly widespread in insect- and plant-associated bacteria. Enzymatic characterization of seven phylogenetically related but distinct ITCases exhibited similar activities on six selected ITCs, suggesting that phylogenetic diversity does not determine the substrate specificity of ITCases. X-ray crystallography studies of two ITCases sharing 42% amino acid sequence identity revealed a highly conserved tertiary structure. Notable features of ITCase include a hydrophobic active site with two Zn ions coordinating water/hydroxide, and a flexible cap that is implicated in substrate recognition that covers the active site. This report reveals the function and structure of the previously uncharacterized family of isothiocyanate hydrolases within the otherwise relatively well-studied superfamily of metallo-β-lactamases.: This study explores a newly discovered protein in the beta-lactamase superfamily: SaxA, or isothiocyanate hydrolase. Isothiocyanates are defensive compounds found in many cabbage-related crop plants, and are currently being investigated for their antimicrobial and anticarcinogenic properties. We show that isothiocyanate hydrolases are responsible for the breakdown of several of these plant defensive chemicals and suggest their potential for mitigating the beneficial effects of isothiocyanates in crop protection and cancer prevention.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase

“…The ITCases belong to the MBL enzyme superfamily. Five MBL proteins of S. sclerotiorum (SS1G_12040, SS1G_11053, SS1G_01079, SS1G_14439 and SS1G_12145) were selected for further study based on their conserved domain structures and sizes 21 . The amino acid sequence identity among these five MBL proteins was lower than 15%, and a phylogenetic analysis together with MBL proteins from other closely related fungal species (and characterized ITCases from bacteria as outgroups) showed that each S. sclerotiorum candidate formed a separate cluster together with their putative homologs from related fungal species.…”

“…Instead, the hydrolysis of ITCs to their corresponding amines and acetamides seems to represent a more important metabolic process. The hydrolysis of ITCs to amines had been previously described in plant pathogenic bacteria, insect gut-associated bacteria and the flea beetle, Psylliodes chrysocephala 18,19,21,22 , but not until now in fungi. Interestingly GL-producing plants themselves are also reported to generate GL-derived amines 7 , albeit via a very different pathway using mercapturic acid pathway products instead of the ITC hydrolytic pathway described in microbes 22 .…”

“…The ITCase-encoding gene SaxA ("survival in Arabidopsis extracts") was first reported in virulent Pseudomonas syringae and shown to be required for bacterial resistance to ITCs 18 . Subsequently, SaxA was found in some gutassociated bacteria from herbivores feeding on Brassicales plants, where it was proposed to facilitate tolerance of host larvae toward dietary ITCs 20,21 . Recently, an amine and its corresponding acetylated derivative (acetamide) were detected as minor products of ITC metabolism in the flea beetle Psylliodes chrysocephala 22 , but it is not known whether this conversion was performed by the insect itself or by its associated microbiota.…”

Functional Variation in Dipteran Gut Bacterial Communities in Relation to Their Diet, Life Cycle Stage and Habitat

“…Several Gammaproteobacteria that can detoxify phenylethyl-isothiocyanate were isolated from the guts of cabbage root fly larvae [48]. Several of these bacteria possess a saxA gene encoding for a hydrolase that can break down various plant-produced isothiocyanates [49]. Deletion of the saxA gene in one of the gut microbes, Pectobacterium carotivorum, prevented this bacterium from being able to degrade plant material [38].…”

Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa

“…Last but not least, the root flies themselves bring along microbial communities in their guts, which may help them to overcome their host plant's chemical defenses (Welte et al, 2016). Some of these gut microbes, for example Pectobacterium spp, are also root pathogens in Brassica crops (van den Bosch et al, 2018). Taken together, it is very likely that the responses triggered by root herbivory are partly triggered by and targeted to microbial pathogens (Sellam et al, 2007).…”