PMC

Comparison of HSS and related structures. A, aspartate dehydrogenase (PDB: 2dc1). B, homospermidine synthase (PDB: 2ph5). C, lysine 6-dehydrogenase (2z2v). D, saccharopine reductase (PDB: 1e5q). The NAD⁺ and NADPH cofactors are shown in black.

Polyamine-related gene clusters/potential operons containing hss orthologues. ODC, alanine racemase-fold ODC; AAT-fold ODC, aspartate aminotransferase fold ornithine decarboxylase; pyruvoyl ADC, arginine decarboxylase. GenBank protein accession numbers are shown for each ORF. a.a., amino acids.

HPLC analysis of polyamines in C. crescentus and A. variabilis. Cells were harvested at stationary phase (1 day for C. crescentus grown in M2G minimal medium and 14 days for A. variabilis grown in light in BG-11 minimal medium). A, C. crescentus CB15; B, A. variabilis ATCC29413. a, fluorescent label; b, sym-homospermidine; c, diaminononane (internal standard).

Polyamine profile of B. japonicum and R. leguminosarum. A, structures of diamine and triamine polyamines. B, HPLC analysis of polyamines in cell extracts of B. japonicum grown to early stationary phase (outer diameter = 0.4; 9 days) in AMS minimal defined medium. C, HPLC analysis of cell extracts of R. leguminosarum, grown to stationary phase (outer diameter = 0.8; 4 days) in AMS defined minimal medium. a, diaminopropane; b, putrescine; c, unknown peak; d, fluorescent label; e, homospermidine; f, diaminoheptane (internal standard).

HPLC analysis of cell extracts from E. coli expressing recombinant HSS orthologues. In each case, the extract from cells expressing the induced homospermidine synthase orthologue is shown in red, and the empty vector control extract is in black. A, B. japonicum/pET21a; B, P. tetraurelia/pET21a; C, Ralstonia phage φRSL1/pET21b; D, S. marylandensis/pET21b; E, O. terrae/pET21b; F, GOS marine metagenome/pET21b. a, diaminopropane; b, putrescine; c, cadaverine; d, fluorescent label; e, spermidine; f, homospermidine; g, unknown peak (shown to be N¹-aminobutylcadaverine).

The structure of the HSS monomer (PDB: 2ph5), indicating regions highly conserved with carboxy(nor)spermidine dehydrogenase. Upper panel, ribbon structure of HSS monomer. The four regions highly conserved with carboxyspermidine dehydrogenase are indicated by the numbers 1–4 (regions shaded dark blue), which refer to the four regions indicated in the alignment between carboxynorspermidine dehydrogenase and HSS sequences shown in supplemental Fig. S5. These four regions all interact with the NAD⁺ cofactor. Lower panel, a slice through HSS showing the deep cleft between the N- and C-terminal domains. The nicotinamide ring of the NAD⁺ cofactor can be seen buried deep within the cleft.

Neighbor-joining tree of HSS orthologues. The unrooted tree was built using PAUP* (as described in Lee et al. ()) with percentage values from 1000 bootstrap replicates indicated; values less than 50% are not shown. The alignment on which the tree is based is presented in supplemental Fig. S1. Only representative α-Proteobacteria are shown (a more complete representation is shown in supplemental Fig. S2). Orthologues with four red asterisks were characterized in this study, and those with one asterisk were characterized previously.

Homospermidine is required for normal growth in R. leguminosarum. A, the effect of the specific ODC inhibitor α-DFMO on growth of R. leguminosarum in minimal defined medium, monitored by optical density. Error bars in panels A–C and E indicate S.E. B, quantified polyamine content of R. leguminosarum cells grown in 5 mm α-DFMO-containing minimal medium and sampled after 24 and 48 h of growth. C, growth of R. leguminosarum in minimal medium containing: black circles, control cells; black squares, 5 mm α-DFMO; asterisks, α-DFMO and 1 mm cadaverine; white circles, α-DFMO and 1 mm norspermidine; white squares, α-DFMO and 1 mm spermidine; black triangles, α-DFMO and 1 mm putrescine. D, polyamine profile, detected by HPLC, of R. leguminosarum cells grown in minimal medium containing 5 mm α-DFMO and 1 mm putrescine (PUT), norspermidine (NSPD), spermidine (SPD), or cadaverine (CAD) with a peak R representing the labeling reagent and a peak of homospermidine (HSPD). E, growth of R. leguminosarum cells in minimal medium containing: black circles, control cells; black squares, 5 mm α-DFMO; black triangles, 5 mm α-DFMO and 0.5 mm homospermidine; white circles, 5 mm α-DFMO and 0.5 mm spermidine; white squares, 5 mm α-DFMO and 0.5 mm norspermidine. F, polyamine profile, detected by HPLC, of R. leguminosarum cells grown with: black trace, control cells; gray trace, 5 mm α-DFMO; red trace, 5 mm α-DFMO and 0.5 mm norspermidine; green trace, 5 mm α-DFMO and 0.5 mm spermidine; blue trace, 5 mm α-DFMO and 0.5 mm homospermidine. DAP, diaminopropane; PUT, putrescine; R, labeling reagent; NSPD, norspermidine; SPD, spermidine; HSPD, homospermidine.

Recurrence of the GAPDH-like fold in the aspartate, arginine, and polyamine pathways. The metabolic routes for lysine (diaminopimelate and α-aminoadipate pathway) and arginine biosynthesis are represented along with bifurcating pathways that stem from various metabolic intermediates (NAD biosynthesis from aspartate, threonine/methionine biosynthesis from aspartate β-semialdehyde, and polyamine biosynthesis from putrescine/aspartate β-semialdehyde). Enzymes with similar folds are indicated with similarly colored circles, and pathways with similar consecutive enzymes that are presumed to stem from a common ancestral pathway () are boxed in dashed lines. Genes encoding GAPDH-like folds are labeled in red and numbered according to three general groups of structural similarity. Group 1 folds display minimal evolutionary cores with few secondary structure elaborations. Group 2 folds include secondary structure elaborations similar to those found in GAPDH. Group 3 folds include different secondary structure elaborations that are common to HSS-like enzymes. The ddh GAPDH-like inserted domain exhibits an unusual topology that is distinct from the three groups and is therefore not numbered. The abbreviations are: argC, N-acetyl-γ-glutamylphosphate reductase; AspDH, aspartate dehydrogenase; hom, homoserine dehydrogenase; dapB, dihydrodipicolinate reductase; lysY, N-acetyl-aminoadipate semialdehyde dehydrogenase; LysDH, l-lysine dehydrogenase; sdh, saccharopine dehydrogenase.