Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69 mM, while its Vmax was 10.61 μkat mg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed.
Carbohydrate sulfotransferases (CHSTs) play a vital role in the production of sulfated polysaccharides (SPs) in algae by catalyzing the sulfation of carbohydrate moieties through the transfer of a sulfuryl group from the donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the present study, putative algal CHSTs with a PF00685, PF03567. PF06990 and PF13469 domain were identified by HMMER search and Protein Basic Local Alignment Search Tool (BLAST) using the well-characterized human CHSTs as queries. Approximately half of the algal CHSTs that contained a PF00685 domain also possessed a PF13469 domain in an overlapping region. These CHSTs were structurally and phylogenetically distinct from algal CHSTs containing PF03567 or/and PF06990 domains. The PF00685/PF13469 domain is commonly found in Chlorophyta, while PF03567 and PF06990 domains are more prevalent in red algae and brown algae, respectively, reflecting the different types of SPs produced by these distinct phyla. Our phylogenetic analyses of algal CHSTs support the hypothesis of a polyphyletic origin, suggesting complex evolutionary histories involving both lineage-specific evolution and significant horizontal gene transfer (HGT) events between algae and organisms from other diverse taxa, including bacteria. In addition, the specificities of algal CHSTs for different carbohydrate moieties and site-specific sulfation patterns were inferred from the phylogenies of human CHSTs and the CHSTs from of algae with known SPs and chemical structures. This approach helps us to bridge the gap in knowledge, as a limited number of algal CHSTs have been biochemically characterized experimentally.