Phenylalanine 4-Hydroxylase Contributes to Endophytic Bacterium Pseudomonas fluorescens’ Melatonin Biosynthesis

Abstract

Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.

Enzyme Promiscuity in Serotonin Biosynthesis, From Bacteria to Plants and Humans

“…Since chloroplasts are the main localization of plant SNAT, and have been hypothesized to be originated from cyanobacteria, the melatonin synthetic pathway in plants is thought to be partially inherited from this group of bacteria ( Tan et al, 2016 ). Consistently, melatonin production via 5-MT was also detected in P. fluorescens , although further research on bacterial ASMT and SNAT genes is needed to fully understand how these reactions really occur ( Jiao et al, 2021 ).…”

“…Alternatively, melatonin can also be synthetized via 5-methoxytryptamine in plants ( Tan et al, 2016 ). Light blue dashed arrows represent putative serotonin and melatonin biosynthetic pathways for bacteria - tryptophan conversion into serotonin via 5-hydroxytryptophan, through phenylalanine hydroxylase (PheH; EC 1.14.16.1; Lin et al, 2014 ; Jiao et al, 2021 ) and bacterial AAAD activity ( Koyanagi et al, 2012 ), and serotonin conversion into melatonin via 5-methoxytryptamine or N -acetylserotonin, through bacterial ASMT and SNAT enzymes ( Tan et al, 2016 ; Ma et al, 2017 ; Jiao et al, 2021 ). Alternatively, tryptophan conversion into tryptamine by bacterial tryptophan decarboxylase (TrpD; EC 4.1.1.105) has also been detected before.…”

“…Indeed, this enzyme has been previously confirmed to hydroxylate both phenylalanine and tryptophan in P. fluorescens ( Lin et al, 2014 ) and the corresponding gene has been detected in multiple bacteria species (contrarily to tryptophan hydroxylase genes, which have not been identified in bacteria so far). Recently, in a follow-up study, the phenylalanine hydroxylase encoding gene from P. fluorescens RG11 was cloned and expressed ( Jiao et al, 2021 ), and the corresponding enzyme also exhibited hydrolase activity with tryptophan as substrate, to form the serotonin precursor 5-HTP. Additionally, the team tested serotonin and melatonin production in a mutant strain lacking functional PheH, in which serotonin and 5-HTP production decreased considerably.…”

“…Microbial enzymes also lack an N -terminal extension of about 200 amino acids, as well as the C-terminal domain, which is involved in tetramerization in the eukaryotic enzymes ( Zhao et al, 1994 ). Most of the AAAH bacterial genes discovered to date are annotated as phenylalanine hydroxylase genes ( Jiao et al, 2021 ). AAAH genes from Pseudomonas aeruginosa ( Zhao et al, 1994 ), Chromobacterium violaceum ( Chen and Frey, 1998 ), Colwellia psychrerythraea ( Leiros et al, 2007 ) and multiple Chlamydia species ( Abromaitis et al, 2009 ) had homology with mammalian hydroxylases.…”

“…However, the affinity of these enzymes for tryptophan is generally lower than that for phenylalanine. Protein engineering studies have shown that AAAH substrate preference can be shifted through substitution of just a small number of residues in the enzyme amino acid sequence, specifically residues that interact with enzyme cofactors and substrates ( Jiao et al, 2021 ). Putative PheH from Pseudomonas aeruginosa , Pseudomonas putida , Pseudomonas fluorescens , Ralstonia eutropha and Xanthomonas campestris have shown small activity with tryptophan, although their substrate preference was stronger for phenylalanine.…”