FIG. 6.

In silico analysis of EctD. (A) Partial alignment of EctD and related proteins. The amino acid sequence of the EctD protein cloned from Chromohalobacter salexigens (Csa1) was used as a query for PSI-BLAST. The sequences used were found in Streptomyces chrysomallus (Schr, [ThpI]), C. salexigens (Csa1 [EctD] and Csa2), Marinobacter aquaeoli (Maq1 and Maq2), Nocardia farcinica (Nfar), Brevibacterium linens (Blin), Bacillus clausii (Bcla), Nitrosococcus oceani (Noce), Sphingopyxis alaskensis (Sala), “Methylobacter alcaliphilus” (Malc), Bordetella parapertussis (Bpar), Alkalilimnicola ehrlichei (Aehr), Dactylosporangium sp. (Dact), Bradyrhizobium japonicum (Bjap), and humans (Huma). Identical and conserved residues are shown against black and gray backgrounds, respectively. The Fe(II)-binding and other residues involved in the active site of the human PhyH protein are indicated by arrows below the sequence. The 2-oxoglutarate-binding residues of PhyH are indicated by dots. Residues that are strictly conserved in all aligned sequences are indicated by asterisks below the PhyH sequence. Residues that are strictly conserved in all proposed ectoine hydroxylases are indicated by arrowheads above the ThpD sequence. The regions used to design the primers for isolation of the C. salexigens ectD gene are indicated by horizontal arrows above the ThpD sequence. (B) Cladogram of the alignment of the complete sequences of the proteins shown in panel A. Sequences in the neighbor-joining tree are identified by their species names and accession numbers. The ectoine hydroxylase family members are boxed to highlight their distinctness from other Fe(II)- and 2OG-dependent enzymes. Bar, 0.2 change per site.