Identification And Characterization Of Bacterial Enzymes Involved In Anaerobic Metabolism Of Aromatic Amino Acids

Phenylalanine,tyrosine and tryptophan are the three aromatic amino acids found in natural proteins.Secondary metabolites during the synthesis and metabolism of these amino acids are also important in lives across the kindoms.Hydroxyphenylacetate decarboxylase(HPAD),an oxygen sensitive glycyl radical enzyme(GRE),was identified in 2001 and was believed to be involved in tyrosine fermentation.It has long been believed that animal feces are malodorant due to the presence of skatole(3-methylindole),a microbial fermentation product of tryptophan.However,the enzyme responsible for the formation of skatole has not been identified.We hypothesized an oxygen sensitive GRE similar to HPAD to be the long sought indoleacetate decarboxylase(IAD).Therefore,we performed comparative genomic studies to search for IAD.A candidate GRE in Olsenella scatologenes was discovered and the recombinant protein was obtained by heterologous expression in E.coli and tested for its catalytic activity.Results demonstrated that the recombinant enzyme is kinetically competent in converting indoleactate to skatole.Further bioinformatic studies identified a dozen of anaerobic bacteria containing this IAD including a human gut bacterium and a bacterium living in the crevice of human mouth quilting.The presence of IAD in the mouth microbiome established the link to halitosis.To investigate the possible mechanism of IAD,a homology model was constructed for Os IAD using Cs HPAD as a template,followed by docking of the indoleacetate substrate.The model suggested that indoleacetate is bound in a similar conformation as hydroxyphenylacetate in Cs HPAD: the acetate group has almost the same conformation,and the indole ring is more or less in the same plane as the phenol ring.The Os IAD residue His514,which is conserved in IAD but not in HPAD,could form a hydrogen bond with the indole N-H.However,given the low homology between the modelled protein and the template,further structural studies are required and are currently underway.Some anaerobic bacteria including human gut bacteria contain HPAD and the product formed by HPAD,cresol is associated with autism.However,how tyrosine is converted to hydroxyphenylacetate is still unknown.We examined the HPAD genomic neighborhood in Clostridium scatologenes and proposed a novel biochemical pathway requiring a transaminase,a hydroxyphenylpyruvate oxidoreductase,a hydroxyphenylacetate Co A phosphate transferase and a hydroxyphenylacetate kinase to convert tyrosine stepwise to hydroxyphenylacetate,which then serves as the substrate of HPAD.Recombinantly produced enzymes were shown catalytic activities,consistent with our hypothesis.Enzyme kinetics and substrate specificities were also measured in order to demonstrate the physiological relevance of these enzymes.Our discovery of IAD has great impact on livestock industry and human health,because skatole has been shown a pneumotoxin and carcinogen to animals and humans.It is also an oviposition attractant that contributes to the propagation of insect-borne human diseases.The characterization of this entire pathway from tyrosine to cresol provides the physiological siginificance of why bacteria harbor the three GREs including HPAD,IAD and Phd B(discovered by another group very recently): to assimilate nitrogen,to generate ATP and to regulate the cytoplasmid p H(decarboxylation generates a carbon anion and depletes a proton).In this pathway,the conversion of tyrosine to hydroxyphenylacetate is coupled to the generation of ATP and reduced ferredoxin,and is thermodynamically reversible.Thus the involvement of HPAD,IAD and Phd B in the final and irreversible step suggests a possible role in providing a net thermodynamic driving force to pull the pathway forward,providing a metabolic rationale for formation of these toxins.