Indoleacetate decarboxylase is a glycyl radical enzyme catalysing the formation of malodorant skatole

Abstract

Skatole is a malodorous compound that contributes to the characteristic smell of animal faeces. Although skatole has long been known to originate from bacterial tryptophan fermentation, the enzyme catalysing its formation has so far remained elusive. Here we report the use of comparative genomics for the discovery of indoleacetate decarboxylase, an O2-sensitive glycyl radical enzyme catalysing the decarboxylation of indoleacetate to form skatole as the terminal step of tryptophan fermentation in certain anaerobic bacteria. We describe its biochemical characterization and compare it to other glycyl radical decarboxylases. Indoleacetate decarboxylase may serve as a genetic marker for the identification of skatole-producing environmental and human-associated bacteria, with impacts on human health and the livestock industry.

Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene‐Producing Glycyl Radical Enzyme

“…In this study, we used sitedirected mutagenesis to replace the Cys‐loop Gln residue in PhdB with a Glu residue (Q484E), which resulted in complete loss of activity; similarly, the Q484A and Q484L mutants showed negligible activity (Figure B). The result for Q484E provides a stark contrast with the other two known GRE decarboxylases, p ‐hydroxyphenylacetate decarboxylase (CsdBC/HpdBC) and indoleacetate decarboxylase (IAD), both of which contain a Glu (rather than Gln) residue in this Cys‐loop position (Figure A). This is an indication that PhdB uses a different catalytic mechanism than these other GRE decarboxylases.…”

“…Notably, the SEC results indicate that wild‐type PhdB occurs as a functional homodimer in solution, as the molecular weight estimated by SEC was approximately 200 kDa and the calculated, sequence‐based value is 95.6 kDa (accounting for the N‐terminal GH scar; see Experimental Section). Other single‐subunit GREs that have been characterized also occur as homodimers …”

“…Phenylacetate decarboxylase (PhdB), which was recently discovered via activity‐guided metaproteomic studies of toluene‐producing microbial communities, has promise as a catalyst for first‐time biochemical synthesis of toluene from renewable resources. PhdB represents one of ten glycyl radical enzyme (GRE) reaction types that have been discovered to date, namely, pyruvate formate‐lyase (EC 2.3.1.54), anaerobic ribonucleotide reductase (EC 1.17.4.1), benzylsuccinate synthase (EC 4.1.99.11), p ‐hydroxyphenylacetate decarboxylase (EC 4.1.1.83), B 12 ‐independent glycerol (and propane‐1,2‐diol) dehydratase (EC 4.2.1.30), choline trimethylamine‐lyase (EC 4.3.99.4), and the very recently discovered (since 2017) trans ‐4‐hydroxy‐ l ‐proline dehydratase, phenylacetate decarboxylase, indoleacetate decarboxylase, and isethionate sulfite‐lyase …”

“…There are three known GRE decarboxylases: p ‐hydroxyphenylacetate decarboxylase (aka CsdBC or HpdBC), phenylacetate decarboxylase (PhdB), and indoleacetate decarboxylase (IAD). Noteworthy similarities and differences exist among these three decarboxylases: 1) All three act on aryl acetates derived from aromatic amino acids (the aryl acetates are p ‐hydroxyphenylacetate, phenylacetate, and indoleacetate derived, respectively, from tyrosine, phenylalanine, and tryptophan), but only PhdB acts on an aromatic moiety that is not substituted with the electronegative elements O or N; 2) There is a single subunit type for PhdB and IAD (for Clostridium scatologenes ), but large and small subunits for CSD/HPD; and 3) The three decarboxylases are each very specific to their cognate substrates, and do not use each other's substrates. For example, IAD does not use phenylacetate or p ‐hydroxyphenylacetate, PhdB does not use p ‐hydroxyphenylacetate, and CsdBC does not use phenylacetate …”

Bioconversion of Biologically Active Indole Derivatives with Indole-3-Acetic Acid-Degrading Enzymes from Caballeronia glathei DSM50014

“…Finally, no intermediate has been proposed which could appear during the conversion of DOAA into catechol. Recently, an enzymatic decarboxylation of IAA to skatole by indoleacetate decarboxylase (Iad) has been identified [32] adding a new metabolic pathway to the IAA catabolism.…”