The reaction mechanism of CDMS (caldomycin synthase) differs from that of exiting homospermidine synthase
We have previously reported that homospermidine is produced as the major polyamine in a gene-disrupted strain for the triamine/agmatine aminopropyl transferase (TtSpeE/TAAPT), a key enzyme for the production of polyamines possessing an aminopropyl moiety in T. thermophilus24. Therefore, it is possible that this bacterium possesses a homospermidine biosynthetic pathway different from the pathways for synthesis of other long and branched polyamines. The DHS gene homolog (TTHA1570) was found in the T. thermophilus genome. It is known that the functional properties of HSS are similar to that of DHS (Fig. 1A), although the number of amino acids and substrate recognition are different from depending on organisms. Thus, the gene of DHS homolog which is the putative polyamine biosynthetic enzyme is named as cdms. We generated an alignment of the highly conserved HSS, DHS and DHS-like HSS (SpeY) amino acid sequences from T. thermophilus, human, Arabidopsis thaliana, Cylindrospermum stagnale and Senecio vernalis using Clustal Omega (Supplemental Fig. 1). The sequences of CDMS showed 27 to 33% identity with the enzymes of other organisms. The lysine residue that is critical for the catalytic activity of DHS is conserved in these proteins28.
To investigate whether CDMS is involved in homospermidine biosynthesis, we constructed a cdms gene-disrupted strain of T. thermophilus. The intracellular polyamine content of the wild-type and gene-disrupted (Δcdms) strains of T. thermophilus grown in minimum medium was analyzed by HPLC (Fig. 2). Peaks corresponding to homospermidine (44) and homospermine (344) were undetectable in the Δcdms strain. This suggests that CDMS is essential for synthesis of homospermidine, and that homospermine (344) is synthesized from homospermidine (44) but not from spermidine (34).
Two types of HSS have been identified in bacteria and plants (Figs. 1B and 1C)14,16. Both types of HSS catalyze the NAD+-dependent transfer of an aminobutyl group from spermidine or putrescine. CDMS was purified to determine its enzymatic properties. As shown in Fig. 3A, a single prominent band with an apparent molecular mass of 38 kDa was obtained by SDS-PAGE. The enzymatic activity of CDMS was studied by adding possible substrate polyamines putrescine, spermidine, or agmatine and combinations of these as substrates (Fig. 3B). Surprisingly, CDMS did not affect levels of putrescine and spermidine (Fig. 3B, lanes 1, 4 and 5) but reduced the level of agmatine (Fig. 3B, lanes 2, 3 and 6) suggesting that agmatine was used as the sole substrate. However, in these assays when agmatine was included as the substrate for CDMS, homospermidine was never detected as a product. Thus, if CDMS is involved in homospermidine synthesis in vitro, there is presumably an intermediary product between agmatine and homospermidine, synthesized by CDMS that serves as the substrate for synthesis of homospermidine.
Identification of reaction product by CDMS
Agmatine was the only identified substrate for CDMS. The results suggest that the first step in homospermidine biosynthesis is a condensation reaction of agmatine. Such a reaction would produce an agmatine derivative, 1,9-bis(guanidino)-5-aza-nonane. However, this guanidyl compound cannot react with o-phthalaldehyde and cannot be detected using the same HPLC method used to identify polyamines and polyamine derivatives such as spermine, spermidine, and homospermidine. Thus, a different fluorescent reagent, benzoin29, which interacts with guanidyl compounds, was used to try to detect 1,9-bis(guanidino)-5-aza-nonane after HPLC separation of the reaction products. As shown in Fig. 4A, peaks corresponding to agmatine (G4) and the putative product of CDMS, “X” were observed after incubation of agmatine with CDMS. The product X is likely 1,9-bis(guanidino)-5-aza-nonane formed via agmatine coupling. To determine if this is the case, synthetic 1,9-bis(guanidino)-5-aza-nonane was compared with product X by HPLC. As shown in Fig. 4B, synthetic 1,9-bis(guanidino)-5-aza-nonane eluted with the same retention time as product X. When the enzymatic reaction included synthetic 1,9-bis(guanidino)-5-aza-nonane as well as the substrate agmatine, the peak area of product X was increased but the retention time was unchanged (Fig. 4C). The results indicate that product X was indeed 1,9-bis(guanidino)-5-aza-nonane, and is named as caldomycin (CDM). This is the first report to show the natural occurrence of caldomycin.
New homospermidine biosynthesis from agmatine by caldomycin synthase (CDMS) together with aminopropylagmatine ureohydrolase (TtSpeB ap ) in T. thermophilus
To confirm that CDMS converts agmatine to caldomycin and that TtSpeBap uses caldomycin as a substrate to produce homospermidine, we purified CDMS and TtSpeBap and carried out in vitro enzymatic reactions followed by HPLC analysis. As shown in Fig. 5, the combination of CDMS and TtSpeBap with agmatine (G4) produced homospermidine (44). In addition, a new intermediate compound was detected as a minor component. The elution time suggested that the new compound is N1-aminobutylagmatine (G44). The structure of the intermediate was confirmed by comparing its elution time with synthetic N1-aminobutylagmatine.
The enzymatic activities of CDMS for substrates such as agmatine, diaminopropane, putrescine, norspermidine, and spermidine revealed that the enzyme has transferase activity as shown in Supplemental Table 1. These results indicated that CDMS utilizes agmatine as the only acceptor in its agmatinyl group transfer reaction (Km; 0.095 ± 0.006 mM, Vmax; 1.07 ± 0.05 µmol/min/mg). Therefore, the enzyme was named as caldomycin synthase (CDMS). In addition, the enzymatic activities of TtSpeBap and TtARG for caldomycin revealed that the two enzymes have ureohydrolase activity as shown in Table 1. In particular, TtSpeBap has very high ureohydrolase activity against caldomycin. In in vivo studies of T. thermophilus, caldomycin and N1-aminobutylagmatine were undetectable, suggesting that TtSpeBap rapidly converts caldomycin to homospermidine in vivo.
The rate of the reaction for CDMS in the presence of a smaller amount of TtSpeBap was significantly increased by temperature elevation from 40oC to 65oC (Supplemental Fig. 2A). The effect of pH on enzymatic activity was investigated over pH range of 6.0 to 10.5 at 65oC (Supplemental Fig. 2B). CDMS showed highest activity at around pH 8.5. We previously reported that homospermidine is increased in T. thermophilus grown at relatively lower temperature such as 60oC, but there is a minor component in the cells grown at the optimal growth temperature such as 70oC. In addition, cell growth and polyamine contents of the polyamine-deficient strain (ΔTtspeA) were recovered when agmatine was added to the culture medium at 60oC23. Especially, homospermidine levels increased together with homospermine. These results indicate that there is a good correlation between the substrate and temperature at which CDMS acts efficiently and the polyamine composition in T. thermophilus.
The catalytic mechanism of CDMS is highly homologous to that of the human DHS
The structures of human DHS (HsDHS)30 and Blastochloris viridis HSS (BvHSS)31 have been determined by X ray crystallography, providing insights into their catalytic mechanisms. To locate the agmatine binding site and possible catalytic mechanism of CDMS, we compared the crystal structures of HsDHS and BvHSS with a structural model of a CDMS-substrate complex that was constructed using an Alphafold2 prediction model. Based on the differences in substrates and products between CDMS, HsDHS, and BvHSS, it was expected that the structures of CDMS and HsDHS would diverge significantly. However, an overall structure and substrate-binding site of the model of CDMS was very similar to the crystal structure of HsDHS. Figure 6 shows the structure of the substrate-binding site of the complex model of CDMS and agmatine (Fig. 6A), along with those of the HsDHS-spermidine complex (PDB ID 6XXJ) (Fig. 6B)30 and the BvHSS-putrescine complex (PDB ID 4TVB) (Fig. 6C)31. Most of the aligned residues of CDMS and HsDHS are identical or similar, with two exceptions of residues having different properties (Phe204 and Leu286 in CDMS vs Asp243 and Asp316 in HsDHS) (Figs. 6A and 6B). The structural model of a CDMS suggests that Glu293 and Ser67 are responsible for the binding to the guanidyl group of agmatine. A conservation of the putative catalytic lysine (Lys299 in CDMS, corresponding to Lys329 in HsDHS) indicates that the catalytic reaction of CDMS likely proceeds via an enzyme-imine covalent intermediate as has been observed for the HsDHS32,33. The model also suggests that Glu136 and Asp143, which are located at the entrance to the substrate-binding site, are responsible for binding the guanidyl group of a second agmatine substrate. To examine this possibility directly, we are currently investigating the structure of CDMS-substrate complexes using X-ray crystallographic studies.