Discovery of ancestral L-ornithine and L-lysine decarboxylases reveals parallel, pseudoconvergent evolution of polyamine biosynthesis

Abstract

Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5′-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable. Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5′-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable. The polyamine spermidine is found throughout bacteria, archaea, and eukaryotes (1Michael A.J. Polyamines in eukaryotes, bacteria, and archaea.J. Biol. Chem. 2016; 291: 14896-14903Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and phylogenetic evidence indicates that it was synthesized in the Last Universal Common Ancestor (LUCA) (2Weiss M.C. Sousa F.L. Mrnjavac N. Neukirchen S. Roettger M. Nelson-Sathi S. Martin W.F. The physiology and habitat of the last universal common ancestor.Nat. Microbiol. 2016; 1: 16116Crossref PubMed Scopus (412) Google Scholar). At physiological pH, the three amines of spermidine are protonated, and consequently spermidine is positively charged and able to interact with negatively charged macromolecules (3Aikens D. Bunce S. Onasch F. Parker 3rd, R. Hurwitz C. Clemans S. The interactions between nucleic acids and polyamines. II. Protonation constants and 13C-NMR chemical shift assignments of spermidine, spermine, and homologs.Biophys. Chem. 1983; 17: 67-74Crossref PubMed Scopus (60) Google Scholar). In Escherichia coli, 90% of spermidine is noncovalently bound to RNA, with only 3.8% in the unbound form (4Miyamoto S. Kashiwagi K. Ito K. Watanabe S. Igarashi K. Estimation of polyamine distribution and polyamine stimulation of protein synthesis in Escherichia coli.Arch. Biochem. Biophys. 1993; 300: 63-68Crossref PubMed Scopus (100) Google Scholar). Spermidine is strongly associated with ribosome function and translation, and in eukaryotes and most archaea, the aminobutyl moiety of spermidine is transferred to a single lysine residue in eIF5A to eventually form the essential hypusine modification (5Park M.H. Wolff E.C. Hypusine, a polyamine-derived amino acid critical for eukaryotic translation.J. Biol. Chem. 2018; 293: 18710-18718Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Hypusinated eIF5A is required for translation of mRNAs encoding polyproline tracts, but it is also required for normal translation elongation and termination (6Dever T.E. Ivanov I.P. Roles of polyamines in translation.J. Biol. Chem. 2018; 293: 18719-18729Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Furthermore, in many eukaryotes and archaea, spermidine is also required for biosynthesis of long-chain polyamines such as spermine and thermospermine and branched chain polyamines (7Michael A.J. Biosynthesis of polyamines and polyamine-containing molecules.Biochem. J. 2016; 473: 2315-2329Crossref PubMed Scopus (81) Google Scholar). The function of spermidine in bacterial growth ranges from being essential, to being required for normal growth, to being dispensable for normal growth (8Michael A.J. Polyamine function in archaea and Biol. Chem. 2018; 293: Full Text Full Text PDF PubMed Scopus (60) Google Scholar). is the biosynthetic of spermidine Spermidine is synthesized from by decarboxylase and spermidine by and decarboxylase (7Michael A.J. Biosynthesis of polyamines and polyamine-containing molecules.Biochem. J. 2016; 473: 2315-2329Crossref PubMed Scopus (81) Google Scholar). The evolutionary history of spermidine biosynthesis and universal distribution in the of by a of biosynthetic of (7Michael A.J. Biosynthesis of polyamines and polyamine-containing molecules.Biochem. J. 2016; 473: 2315-2329Crossref PubMed Scopus (81) Google Scholar). be synthesized from by arginine decarboxylase from by ornithine decarboxylase The product of arginine agmatine, be to by by the of deiminase and the of the same function from protein also known as nonhomologous of in PubMed Scopus Google Scholar), in emergence of ADC from protein A.J. of biosynthetic J. PubMed Scopus Google Scholar). 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F. F. C. S. of bacterial AAT-fold amino acid decarboxylases and of Biol. 2018; Google that the and are of the acid-inducible forms. this the only known in the ancestral form AAT-fold decarboxylase was the of we to ODC was also present the we to be ADC, ODC, LDC the found in the and are with this most an ADC from the that is the of the and ADC C. K. C. A.J. The bacterial of the polyamine biosynthetic PubMed Scopus Google Scholar). is of the function of the and ancestral form AAT-fold decarboxylase species ancestral form AAT-fold decarboxylase and in is ADC and the it that is in the modification of translation is are also found the AR-fold In the AR-fold decarboxylase activities are by ADC, and decarboxylase (7Michael A.J. Biosynthesis of polyamines and polyamine-containing molecules.Biochem. J. 2016; 473: 2315-2329Crossref PubMed Scopus (81) Google Scholar). The activities A.J. for biosynthesis of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google are the last in lysine acid are in a modification of the translation a residue in the Furthermore, the AR-fold ADC as an ancestral form that is the same as decarboxylases in the and an extended form amino acids containing a and a that the extended ADC a the form of the AR-fold decarboxylases M. A.J. and of arginine decarboxylases in polyamine biosynthesis and essential in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. A.J. of a of amino acid of with for and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a in the AR-fold decarboxylase for the ancestral form AAT-fold decarboxylase The emergence of ancestral ADC, ODC, and LDC from a common in the AAT-fold may be in by the ability of ADC, ODC, and LDC to decarboxylate three amino acid substrates and are in is nascent to the extended form of the ADC, ODC, and LDC with N-terminal the of is of and S. S. M. M. emergence of in Escherichia Chem. Biol. 2018; PubMed Scopus Google Scholar). extended form from the species and are also able to decarboxylate L-lysine and but of less of functionally ornithine in acid Microbiol. PubMed Scopus Google Scholar). coli, biosynthetic forms of both ADC and ODC, the bacterial species that the ancestral form AAT-fold ODC only and the species encoding ADC only the for ODC The S. is an ornithine and arginine and exogenous arginine is an essential amino acid for growth of arginine and in the by and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an and in the is to arginine The arginine deiminase of S. ornithine from but the ornithine is by the C. M. S. of the arginine deiminase by with arginine and of PubMed Scopus Google Scholar). In F. is also an ornithine and arginine S. F. the arginine deiminase this exogenous ornithine produced by the arginine deiminase of S. In the of S. and F. the to by ADC ODC activities to may have by and the of arginine and ornithine in the The ancestral form in are from C. F. F. and C. it is that an for ODC as the by is in the same as F. and F. is also from an The extended form AAT-fold ADC, ODC, and LDC an N-terminal domain from a domain of a response to the of an ancestral form M. A.J. and of arginine decarboxylases in polyamine biosynthesis and essential in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the N-terminal the extended form in in the domain homologous to the ancestral The N-terminal domain and have in the common of the extended form ADC, ODC, and LDC is that the presence of to and protein of the in that the N-terminal domain was the is the have the that the domain The extended form acid-inducible decarboxylase have also a amino that in with the decarboxylase the are also of ADC, ODC, and LDC activities in the ancestral and extended form emergence of ADC, ODC, and LDC activities homologous from the same S. K. S. of activities in the in of PubMed Scopus Google Scholar). the of the ancestral form ADC ODC LDC), ancestral form an and decarboxylate the amino The parallel, emergence of the preferred the homologous ancestral and extended forms an the same to of have form of for LDC in species such as the LDC, in biosynthesis M. A.J. of a lysine decarboxylase in Microbiol. PubMed Scopus (33) Google Scholar), emerged from the decarboxylase the AAT-fold but to the ancestral and extended form ODC and LDC ancestral forms and extended the phylogenetic diversity of the to Fusobacteria, Nitrospirae, Caldiserica, and discovery of the parallel, homologous but of ADC, ODC, and LDC an to emergence of the same activities from homologous of was with of that on of in was to the and The was on for and for of was to the and for of the was on with and containing the decarboxylase in to with of and in and in a was for to and The was to a with and and the from the with a of by was using and protein was using a protein with the and protein The encoding fusion by from of F. and of and and was for for for and for a for The domain of the F. fusion protein was by from using and and by The F. fusion protein and decarboxylase domain the using and S. and ODC in for with by in and in of was to and was to with was for with by and to being in growth in to an of in of and as ODC was by of from as A.J. Polyamine in ornithine decarboxylase ornithine Biochem. PubMed Scopus Google Scholar). in containing and this was of from an The amino acid and was to the and with an on and was to that the the on to a and the of this was as the of the to using the function of the was from to using an of for J. of a for decarboxylase PubMed Scopus Google and an of for in in a in containing L-arginine, and for and by of of the the was to of and by for Two of was for and for The of containing the polyamines was transferred to a and in a chemical as S. J. A.J. for J. PubMed Scopus Google Scholar). was on an using an that was to an single in the with a of was a of with a using a with containing by and this was to N-terminal and to from for protein was with with and PubMed Scopus Google using the in a to a phylogenetic by using J. phylogenetic for 2016; PubMed Scopus Google with for a protein and for phylogenetic Biol. 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