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

Liu, Dazhi, Wei, Yifeng, Liu, Xuyang, Zhou, Yan, Jiang, Li, Yin, Jinyu, Wang, Feifei, Hu, Yiling, Nanjaraj Urs, Ankanahalli N., Liu, Yanhong, Ang, Ee Lui, Zhao, Suwen, Zhao, Huimin, Zhang, Yan·2018·Nature Communications
DOI 10.1038/s41467-018-06627-xPMC PMC6181972
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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 O 2-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.

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Fig. 1 Pathways for fermentation of aromatic amino acids. Tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp) are converted into cresol, toluene, and skatole, respectively. HPAD p-hydroxyphenylacetate decarboxylase, PhdB phenylacetate decarboxylase, and IAD indoleacetate decarboxylase
Fig. 1 Pathways for fermentation of aromatic amino acids. Tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp) are converted into cresol, toluene, and skatole, respectively. HPAD p-hydroxyphenylacetate decarboxylase, PhdB phenylacetate decarboxylase, and IAD indoleacetate decarboxylaseCC BY · PubMed Central
Fig. 2 Maximum likelihood phylogenetic tree of GREs. Included are Cs GREs (red), Os GREs (green), and biochemically validated GREs in other organisms (black). Of the Cs and Os GREs, only CsHPAD has been previously biochemically validated. Proposed functions of the other Cs and Os GREs are given in brackets. Candidate IADs are enclosed in the blue ellipse, of which OsIAD was validated in this study. PFL pyruvate formate lyase, TdcE 2-keto acid formate lyase, CutC choline-trimethylamine lyase, PDH propanediol dehydratase, GDH glycerol dehydratase, HypD trans-4-hydroxy-l-proline dehydratase, BssA benzylsuccinate synthase, AssA alkylsuccinate synthase, PhdB phenylacetate decarboxylase, HPAD p-hydroxyphenylacetate decarboxylase, and IAD indoleacetate decarboxylase reported in this study. Bootstrap confidence values >50 are indicated on the nodes
Fig. 2 Maximum likelihood phylogenetic tree of GREs. Included are Cs GREs (red), Os GREs (green), and biochemically validated GREs in other organisms (black). Of the Cs and Os GREs, only CsHPAD has been previously biochemically validated. Proposed functions of the other Cs and Os GREs are given in brackets. Candidate IADs are enclosed in the blue ellipse, of which OsIAD was validated in this study. PFL pyruvate formate lyase, TdcE 2-keto acid formate lyase, CutC choline-trimethylamine lyase, PDH propanediol dehydratase, GDH glycerol dehydratase, HypD trans-4-hydroxy-l-proline dehydratase, BssA benzylsuccinate synthase, AssA alkylsuccinate synthase, PhdB phenylacetate decarboxylase, HPAD p-hydroxyphenylacetate decarboxylase, and IAD indoleacetate decarboxylase reported in this study. Bootstrap confidence values >50 are indicated on the nodesCC BY · PubMed Central
Fig. 3 Genome neighbourhood of IAD and HPAD from Cs and Os. (GenBank accession numbers CP009933 and LOJF01000000 respectively). HPAD p-hydroxyphenylacetate decarboxylase, HPADAE HPAD activating enzyme, IAD indoleacetate decarboxylase, IADAE IAD activating enzyme, MFS major facilitator superfamily transporter
Fig. 3 Genome neighbourhood of IAD and HPAD from Cs and Os. (GenBank accession numbers CP009933 and LOJF01000000 respectively). HPAD p-hydroxyphenylacetate decarboxylase, HPADAE HPAD activating enzyme, IAD indoleacetate decarboxylase, IADAE IAD activating enzyme, MFS major facilitator superfamily transporterCC BY · PubMed Central
Fig. 4 EPR spectra and enzymatic assays of OsIAD. a X-band EPR spectra of IAD reconstituted with IADAE and SAM in the presence or absence of reductant (Ti(III) citrate). b Reaction requirements and substrate specificity of IAD. IAAK, HPAAK, and PAAK are the potassium salts of indoleacetic, p-hydroxyphenylacetic, and phenylacetic acids, respectively. (The error bars represent the standard deviation of three individual experiments.) c Detection of skatole formation in the IAD-catalysed decarboxylation of IAAK using GC-MS. GC-MS elution profiles of authentic standards of skatole, negative controls omitting SAM and IAAK and a complete assay are displayed as labelled. The internal standard 2,3-dimethylindole is included in each sample. d Mass spectrum of the skatole peak
Fig. 4 EPR spectra and enzymatic assays of OsIAD. a X-band EPR spectra of IAD reconstituted with IADAE and SAM in the presence or absence of reductant (Ti(III) citrate). b Reaction requirements and substrate specificity of IAD. IAAK, HPAAK, and PAAK are the potassium salts of indoleacetic, p-hydroxyphenylacetic, and phenylacetic acids, respectively. (The error bars represent the standard deviation of three individual experiments.) c Detection of skatole formation in the IAD-catalysed decarboxylation of IAAK using GC-MS. GC-MS elution profiles of authentic standards of skatole, negative controls omitting SAM and IAAK and a complete assay are displayed as labelled. The internal standard 2,3-dimethylindole is included in each sample. d Mass spectrum of the skatole peakCC BY · PubMed Central
Fig. 5 Multiple-sequence alignments of HPADs and IADs, highlighting key residues. a Thiyl radical loop region containing conserved Cys• and Glu1 residues, highlighted in red and blue, respectively. A His residue possibly involved in the catalytic mechanism of IAD is coloured orange (Cd Clostridium difficile; Tf Tannerella forsythia; Cb Clostridium botulinum; Fc Faecalicatena contorta). b Region containing Glu2, highlighted in green. Glu2 is conserved in HPADs but not in IADs
Fig. 5 Multiple-sequence alignments of HPADs and IADs, highlighting key residues. a Thiyl radical loop region containing conserved Cys• and Glu1 residues, highlighted in red and blue, respectively. A His residue possibly involved in the catalytic mechanism of IAD is coloured orange (Cd Clostridium difficile; Tf Tannerella forsythia; Cb Clostridium botulinum; Fc Faecalicatena contorta). b Region containing Glu2, highlighted in green. Glu2 is conserved in HPADs but not in IADsCC BY · PubMed Central

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Authors
Liu, D., Wei, Y., Liu, X., Zhou, Y., Jiang, L., Yin, J., Wang, F., Hu, Y., Nanjaraj Urs, A. N., Liu, Y., Ang, E. L., Zhao, S., Zhao, H., & Zhang, Y.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-10-11
Volume
9
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-018-06627-x
PMC
PMC6181972
Cited by
63
References
3
Language
en
Licence
Funders
National Natural Science Foundation of China (31570060)

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