Multiple sequence alignment analysis of putative domains of CfNOS and other NOSs deposited in GenBank.

(A) The solid straight line indicated the major domains or binding regions predicted by SMART program, including a PDZ domain (Asp25–Asp100), an NO synthase domain (Gly427–Lys799), a flavodoxin 1 region (Ile843-Trp1018), an FAD (Thr1075-Trp1304) and an NAD binding region (Met1336-Thr1459). (B) The species and the GenBank accession numbers were as follows: Homo sapiens (AAA62405.1), Drosophila melanogaster (AAC46882.1), Aplysiacalifornica (AAK83069.1), Chlamys farreri and Strongylocentrotus purpuratus (XP_003729305.1). The black shadow region indicated positions where all sequences shared the same amino acid residues. Similar amino acids were shaded in grey. Gaps were indicated by hyphens to improve the alignment. The binding sites for heme (Thr488–Asp509), BH₄ (Asn750–Ile782), Ca²⁺/calcium (CaM) (Lys812–Lys840), FMN (Thr847–Lys870, Ser893–Thr906, Ser968-Ser1000), FAD (Ala1118-Asn1129, Gln1261-Ser1270), NADPH (Met1336-Arg1353, Arg1441-Gln1452), and the location of an autoinhibitory loop (Asn922–Leu965) were identified by multiple sequences alignment.