Enantioselective transformation of phytoplankton-derived dihydroxypropanesulfonate by marine bacteria

Abstract Chirality, a fundamental property of matter, is often overlooked in the studies of marine organic matter cycles. Dihydroxypropanesulfonate (DHPS), a globally abundant organosulfur compound, serves as an ecologically important currency for nutrient and energy transfer from phytoplankton to bacteria in the ocean. However, the chirality of DHPS in nature and its transformation remain unclear. Here, we developed a novel approach using chiral phosphorus-reagent labeling to separate DHPS enantiomers. Our findings demonstrated that at least one enantiomer of DHPS is present in marine diatoms and coccolithophores, and that both enantiomers are widespread in marine environments. A novel chiral-selective DHPS catabolic pathway was identified in marine Roseobacteraceae strains, where HpsO and HpsP dehydrogenases at the gateway to DHPS catabolism act specifically on R-DHPS and S-DHPS, respectively. R-DHPS is also a substrate for the dehydrogenase HpsN. All three dehydrogenases generate stable hydrogen bonds between the chirality-center hydroxyls of DHPS and highly conserved residues, and HpsP also form coordinate–covalent bonds between the chirality-center hydroxyls and Zn2+, which determines the mechanistic basis of strict stereoselectivity. We further illustrated the role of enzymatic promiscuity in the evolution of DHPS metabolism in Roseobacteraceae and SAR11. This study provides the first evidence of chirality’s involvement in phytoplankton-bacteria metabolic currencies, opening a new avenue for understanding the ocean organosulfur cycle.

Table S1.Primers of site-directed mutagenesis.

Fig
Fig. S1 1 H NMR of synthetic P-L-Ala.

Fig. S3
Fig. S3 The optimization of RpHpsN structure.(A) Dimer structure of RpHpsN before optimization.(B) Structural alignments of the RpHpsN crystal structure with AlphaFold2predicted RpHpsN and L-histidinol dehydrogenase from E. coli MC1061 (PDB ID: 1KAE).The significant difference in spatial position of domain A is shown in dotted boxes.(C) Structural alignments of domain A of the RpHpsN crystal structure with domain A of L-histidinol dehydrogenase.(D) RpHpsN structure after optimization using AlphaFold2-predicted structure.

Fig. S5
Fig. S5 Intercellular metabolite analysis of R. pomeroyi DSS-3.(A) Extracted ion chromatogram of sulfolactaldehyde (m/z 152.9858) produced from R. pomeroyi DSS-3 utilizing R-or S-DHPS.Cultures of R. pomeroyi DSS-3 utilizing acetate were used as a control group.(B) MS/MS fragmentations of sulfolactaldehyde generated in R-DHPS treatment group (top) and S-DHPS treatment group (bottom).(C) Extracted ion chromatogram of sulfolactate (m/z 168.9798) produced from R. pomeroyi DSS-3 utilizing R-or S-DHPS as carbon source.Cultures of R. pomeroyi DSS-3 utilizing acetate were used as a control group.(D) MS/MS fragmentations of sulfolactate generated in R-DHPS treatment group (top) and S-DHPS treatment group (bottom).

Fig. S6
Fig. S6 Intercellular metabolites analysis of D. shibae DFL 12. (A) Extracted ion chromatogram of sulfolactaldehyde (m/z 152.9858) produced from D. shibae DFL 12 utilizing R-or S-DHPS as carbon source.Cultures of D. shibae DFL 12 utilizing acetate were used as a control group.(B) MS/MS fragmentations of sulfolactaldehyde generated in R-DHPS treatment group (top) and S-DHPS treatment group (bottom).(C) Extracted ion chromatogram of sulfolactate (m/z 168.9798) produced from D. shibae DFL 12 utilizing R-or S-DHPS as sole carbon source.Cultures of D. shibae DFL 12 utilizing acetate were used as a control group.(D) MS/MS fragmentations of sulfolactate generated in R-DHPS treatment group (top) and S-DHPS treatment group (bottom).

Fig. S7
Fig. S7 Analysis of structures and enzymatic properties.(A) Enzymatic activity assay monitoring NADH formation accompanying S-DHPS oxidization by DsHpsP (0.5 μM).The structure alignment of DsHpsP (green) with AlphaFold2-predicted RpHpsP (blue), with a rmsd value of 0.83 Å over 325 residues.The conserved active sites (His59, Glu60 and Glu139) are shown as sticks.(B) Effects of metal ions on the enzymatic activity of DsHpsP and RpHpsN.

Fig
Fig. S8 Extracted ion chromatograms of candidate cysteinolic acid (m/z 156.0325) extracted from DHPS-producing diatoms and coccolithophores.Corresponding MS next to the chromatograms in the right panel.RT, retention time.

Fig. S14
Fig. S14 Structural difference between RpHpsO, DsHpsP, and RpHpsN compared to their homologs.Severely non-conserved regions are colored in red and are shown as sticks.

Table S2 .
Data collection and final refinement for crystals of RpHpsO, DsHpsP, and RpHpsN.Statistics for the highest-resolution shell are shown in parentheses.

Table S5 .
The correlation analysis among gene expression from Roseobacteraceae and SAR11 in Tara Oceans with environmental factors and algae.