## 3 Results
### Bioinformatic Analysis and the Molecular Modeling of EtABC\({}_{S. salarius}\)  
Multiple sequence alignments were carried out with ClustalX and ESPriP 3.0 [31; 32] (Figures S1-S3). The result showed that EtAt\({}_{S. salarius}\), EtB\({}_{S. salarius}\) and EtC\({}_{S. salarius}\) respectively belonged to the acetyltransferase (GNAT) family (PF00583) [33], aminotransferase class-III family (PF00202) [34] and ectoine synthase family (PF06339) [35], which had been identified and characterized from various species of microorganism. As shown in Tables S1-S3, EtA\({}_{S. salarius}\), EtB\({}_{S. salarius}\) and EtC\({}_{S. salarius}\) from _S. salarius_ 1A01339 shared high identities with widely used ectoine enzymes from _H. elongata_ DSM 2581, as 83.3% identities with EtC\({}_{H. elongata}\), 85.7% identities with EttB\({}_{H. elongata}\) and 73.6% identities with EtC\({}_{H. elongata}\), respectively. Furthermore, we also determined the phylogenetic relationships between ectoine synthesis enzymes and other reviewed proteins from UniProtKB database. As shown in Figures S4-S6, the phylogenetically closest proteins to EtAt\({}_{S. salarius}\) were the EtAt from _Halomonas elongata_ DSM 2581 and _Chromolabolacter salexigens_ DSM 3043, as 83.3% identities. While EttB from _Halomonas elongata_ DSM 2581 and _Chromolobacter salexigens_ DSM 3043 had closest relationship with EtB\({}_{S. salarius}\) and shared highest identities of 85.7% and 84.8%. Similarly, EtC from _Halomonas elongata_ DSM 2581 and _Chromolobacter salexigens_ DSM 3043 had closest relationship with EtC\({}_{S. salarius}\) and shared highest identities of 73.6% and 87.7%, respectively. These results proved that EtAt\({}_{S. salarius}\), EtB\({}_{S. salarius}\) and EtC\({}_{S. salarius}\) could be the functional proteins involved in the ectoine synthesis in _S. salarius_ 1A01339 and help the host adapt to the disadvantaged environmental condition. To analyze the molecular structures of the ectoine-synthesis enzymes, three crystal models were predicted and elucidated by using the solved data as template, respectively. As shown in Figure 2, the EtA\({}_{S. salarius}\) protein adopted the classical fold shared in acetyl-transferase family (PF00583), which usually formed an inner parallel/antiparallel twisted \(\beta\)-sheet and four flanked \(\alpha\)-helices. Further inspection revealed that the functional enzyme was assembled as an asymmetric homodimer, in which EtA\({}_{S. salarius}\) monomers were rotated against each other by about 180\({}^{\circ}\) and included an aromatic tyrosine (Tyr60) from the other opposite monomer. Notably, the strictly conserved amino acid residues, Asp55, Tyr60 (other opposite monomer), Trp102, Gln103, His176, and Glu179, formed an architecture of the substrate-binding pocket to contribute to the binding interaction and catalytic reaction with L-2,4-diaminobutyrate (DAB) (Figure S7) [27]. The results of sequence alignments and crystallized analysis showed that \(\text{EctB}_{\text{S. \saluris}}\) model contained eight polypeptide chains forming two functional tetramers, which was a dimer of dimers reported as a member of the class III PLP-dependent aminotransferases (PF0020). Each dimer consisted of two monomers, in which a central region was folded by seven-stranded antiparallel \(\beta\)-sheets core and surrounded by nine \(\alpha\)-helices, and another small region, consisted of three-stranded \(\beta\)-sheets core and surrounded by three \(\alpha\)-helices, was situated on the surface of the monomer (Figure 3). Previous reports revealed that the active site of class III DABA aminotransferases has two binding pockets with conserved recognition residues involved in forming bonds with the neighboring dimer to maintain structural integrity of the tetrameric model, binding PLP to shape the active site, and forming strong directional bonds to the substrate.