Characterization of sulfate assimilation in marine algae focusing on the enzyme 5'-adenylylsulfate reductase

Abstract

5'-Adenylylsulfate (APS) reductase was characterized in diverse marine algae. A cDNA encoding APS reductase from Enteromorpha intestinalis (EAPR) was cloned by functional complementation of an Escherichia coli cysH mutant. The deduced amino acid sequence shows high homology with APS reductase (APR) from flowering plants. Based on the probable transit peptide cleavage site the mature protein is 45.7 kD. EAPR expressed as a His-tagged recombinant protein catalyzes reduced glutathione-dependent reduction of APS to sulfite, exhibiting a specific activity of approximately 40 micromol min(-1) mg protein(-1) and Michealis-Menten kinetic constants of approximately 1.4 mM for reduced glutathione and approximately 6.5 microM for APS. APR activity and expression were studied in relation to the production of 3-dimethylsulfoniopropionate (DMSP), a sulfonium compound produced by many marine algae. A diverse group of DMSP-producing species showed extremely high enzyme activity (up to 400 times that found in flowering plants). Antibodies raised against a conserved peptide of APR strongly cross-reacted with a protein of 45 kD in several chlorophytes but insignificantly with chromophytes. In the chlorophyte Tetraselmis sp., APR activity varies significantly during the culture cycle and does not follow the changes in cellular DMSP content. However, a positive correlation was found between cell-based APR activity and specific growth rate.

Figure 1

Alignment of the deduced E. intestinalis EAPR amino acid sequence with Arabidopsis APR3 (AF01628). The sequence was aligned with PileUp program (Genetics Computer Group, Madison, WI). Putative reductase-domain and Grx-domain are positioned between amino acid numbers 36 to 116 and 117 to 446, respectively. Residues of important to the catalytic function and tertiary structures are indicated with dots, asterisks, or diamonds.

Figure 2

Complementation of E. coli cysH by E. intestinalis APR requires GSH, but not Trx. wt refers to the cysH strain carrying wild-type alleles for trxB, gshA, and gshB. The others are cysH strains carrying the designated mutation. The cultures were incubated for 40 h at 30°C on M9 plate without Cys.

Figure 3

Temperature dependence of APR activity of purified EAPR recombinant protein. A, Arrhenius plot; B, log transformation. APR activity was determined as described in the “Materials and Methods” at the temperatures indicated in the graph.

Figure 4

Comparison of ARS reductase protein antigenicity from marine algae. All of the lanes were loaded with equal amount of protein (6 μg) except for Ulva pertusa (30 μg) and the purified recombinant protein from pET-EAPR (2 ng). Odd-numbered lanes are controls blotted with preimmune antiserum, whereas even-numbered lanes are blotted with APR antiserum. A, EI (E. intestinalis): total activity was 120 pmol min⁻¹; UP (U. pertusa): total activity was 16 pmol min⁻¹; Tetra (Tetraselmis sp.): total activity was 1,300 pmol min⁻¹; Dun (D. salina): total activity was 390 pmol min⁻¹; Pure APR (purified recombinant protein from pET-EAPR): total activity was 90 pmol min⁻¹. B, Tetra (Tetraselmis sp.): total activity was 1,300 pmol min⁻¹; Iso (I. galbana): total activity was 1,480 pmol min⁻¹; Ehux (E. huxleyi): total activity was 1,500 pmol min⁻¹; TW (T. weissflogii): total activity was 1,400 pmol min⁻¹; TO (T. oceanica): total activity was 800 pmol min⁻¹; HT (H. triquetra): total activity was 30 pmol min⁻¹.

Figure 5

APR activity from Tetraselmis sp. cultures grown with abundant N supply (A) and limited N supply (B). Dotted lines indicate the nitrate level in the cultures. Dash lines with small white circles or white squares are growth curves of the cultures indicated by the cell densities of the cultures. Black circles or squares represent APR activities normalized to cellular protein content, whereas white circles or squares represent APR activities based on cells. Each data point was the average of six measurements from four independent cultures.

Figure 6

Correlation of APR activity and specific growth rate (μ). White circles were data points from cultures grown with abundant N supply, whereas white squares were data points from cultures grown with limited N supply. Each data point represents the average of six measurements from four independent cultures.

Figure 7

APR activity and intracellular DMSP content during a growth cycle in batch cultures of Tetraselmis sp. grown under N-sufficient supply (A) and N-limited supply (B) conditions. Cell-based APR activity is indicated as the dashed lines, whereas intracellular DMSP content is indicated as the solid lines with white circles or squares. Dotted lines indicate the nitrate level in the cultures. Each data point was the average of six measurements from four independent cultures.