Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

Abstract

Background: Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II).

Results: Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria.

Conclusions: Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes an exceptional model of acquisition and maintenance of redundant gene functions between non-homologous genes as a result of convergent evolution. Subsequently, although old episodic events of HGT could not be excluded, the results supported a prevalent role of gene loss in explaining the distribution of DXR-II in specific pathogenic eubacteria. Our results highlight the importance of the functional characterization of evolutionary shortcuts in isoprenoid biosynthesis for screening specific antibacterial drugs and for regulating the production of isoprenoids of human interest.

Figure 1

Isoprenoid and amino acid biosynthetic pathways. A) Pathways for the biosynthesis of isoprenoid precursors. On the left, MVA pathway is represented. Enzymes are indicated in bold: AACT, acetoacetyl-CoA thiolase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMVK, 5-phosphomevalonate kinase; DPMD, 5-diphosphomevalonate decarboxylase; IDI, IPP/DMAPP isomerase. On the right, MEP pathway steps are described: GAP, D-glyceraldehyde 3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME,4-diphosphocytidyl-2-C-methyl-D-erythritol; MEcPP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBPP, 4-hydroxy-3-methylbut-2-enyl diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate. Enzymes are indicated in bold: DXS, DXP synthase; DXR, DXP reductoisomerase; MCT, MEP cytidylyltransferase; CMK, CDPME kinase; MDS, ME-cPP synthase; HDS, HMBPP synthase; HDR, HMBPP reductase; IDI, IPP isomerase. B) Amino acid biosynthesis. The common pathway (CP) is highlighted in black and enzymes indicated in bold: AK, aspartokinase; ASDH, aspartate semialdehyde dehydrogenase; HD, homoserine dehydrogenase. Solid arrows indicate single catalytic steps and dashed arrows mark multiple steps.

Figure 2

Phylogeny of DXR-II and DLO related sequences. ML cladogram depicting the evolutionary relationships among 53 DXR-II and 77 related protein sequences. Three clades defining main families are indicated. Statistical support on relevant clades is indicated by values next to nodes (ML aLRT support values/BA posterior probabilities/NJ bootstrap values). Sequence names are colored according to taxonomical groups (see legend). Sequence names include the bacterial strain name, followed by two pairs of square brackets: the first pair encloses the classification of the given bacterial strain according to the distribution of enzymes of the i) MEP and MVA pathways, left side of the vertical bar (i.e. classes A, +MEP pathway enzymes –DXR; B, +MEP pathway enzymes + DXR; C, -MEP + MVA pathway enzymes -DXR; D, +MEP + MVA pathway enzymes + DXR; E, -MEP -MVA pathway enzymes -DXR) and ii) CP pathway, right side of the vertical bar (i.e. A, complete CP pathway; B, incomplete CP pathway –AK_HD). The second pair of brackets represents the INTERPRO protein functional domains found i.e. 1, NAD(P)-binding domain (IPR016040); 2, Aspartate/homoserine dehydrogenase, NAD-binding (IPR005106); 3, Oxidoreductase, N-terminal (IPR000683); 4, Dihydrodipicolinate reductase, N-terminal (IPR000846); 5, Quinate/shikimate 5-dehydrogenase/glutamyl-tRNA reductase (IPR006151); 6, SAF domain (IPR013974). Asterisks indicate sequences for which DXR-II activity was previously tested through complementation assays [23] and Additional file 5.

Figure 3

3D architecture of DXR-II showing relevant and functional divergence residues. A, view of the DXR-II dimer. Chain A is represented as cartoon backbone highlighting secondary structures and chain B as its molecular surface equivalent. B, Close-up view of the active site and of residues participating in substrate/fosmidomycin binding, including position 229, also predicted as related to functional divergence. C, Close-up view of the cavity at the dimer interface highlighting conserved residues involved in interactions between the two subunits of the DXR-II dimer, including position 320, also predicted as related to functional divergence. The N-terminal, central and C-terminal domains are shown in grey, blue and cyan, respectively. Residues predicted as involved in functional divergence of DXR-II are shown in red. Residues identified as involved in dimerization, fosmidomycin/substrate binding and the active site are shown in yellow, violet and green, respectively. The competitive inhibitor fosmidomycin is colored in orange. Molecular graphics were produced with VMD 1.9.1 [29] on the basis of the crystal structure of B. abortus DXR-II (pdb: 3upy) [26].