*2.1. Gene Cloning and Sequence Analysis*

The *psleudh* gene was amplified from genomic DNA of the strain ANT178. It consisted of an ORF of 1209 bp, encoded a protein of 402 amino acid resides with a theoretical p*I* of 5.08. Furthermore, the DNA sequence of *psleudh* was submitted to the GenBank database with the accession number of MH322031. Based on sequences alignment, PsLeuDH showed the highest sequence similarity (88.0%) with LeuDH from *Pseudoalteromonas nigrifaciens* (ASM53600), followed by a sequence similarity of 65.0% with LeuDH from *Colwellia piezophila* (WP\_019029130). More importantly, PsLeuDH had a conserved Phe binding site (I344) and NAD<sup>+</sup> binding sites (G233, G235, T236, V237, D256, I257, A261, C290, A291, C312, and N314). The coenzyme binding domain of NAD<sup>+</sup> in LeuDH was capable of catalyzing the reversible oxidative deamination of L-leucine and several other branched chain amino acids to form the corresponding 2-oxo acid derivatives. This domain could be classified as a member of the Rossmann fold superfamily, comprising a plurality of different dehydrogenases, wherein the amino acid dehydrogenase family comprises a common feature: a beta-sheet-alpha helix-beta sheet conformation [15]. PsLeuDH had this structural feature from Figure 1, further demonstrating that PsLeuDH was a member of the Glu/Leu/Phe/Val dehydrogenase family.

**Figure 1.** Amino acid sequence alignment of PsLeuDH and related LeuDH. PsLeuDH, *Pseudoalteromonas* sp. ANT178 LeuDH (MH322031); PnLeuDH, *Pseudoalteromonas nigrifaciens* (ASM53600); *Cp*LeuDH, *Colwellia piezophila* (WP\_019029130); RsLeuDH, *Rheinheimera salexigens* (WP\_070050751); SbLeuDH, *Shewanella baltica* BA175 (AEG11165); BsLeuDH, *Bacillus sphaericus* ATCC4525 (PDB ID:1LEH); BtLeuDH, *Bacillus thuringiensis* (WP\_001162678); and LsLeuDH, *Laceyella sacchari* (KR065697). , NAD binding site; , Phe binding site.

#### *2.2. Homology Modeling and Analysis of PsLeuDH*

BsLeuDH (PDB ID:1LEH), encoded 364 amino acids, was isolated from mesophilic bacteria *Bacillus sphaericus* ATCC4525 [16], which exhibited the highest sequence identity (51%) to PsLeuDH using DALI server. The comparative analysis of the 3D structure of PsLeuDH and the mesophilic enzyme Bs-LeuDH was shown in Figure 2. It could be seen that two LeuDHs had a similar NAD<sup>+</sup> binding site and Phe binding site.

**Figure 2.** Three-dimensional structure comparison of PsLeuDH and BsLeuDH model. PsLeuDH, tv-blue; BsLeuDH, cyan; NAD<sup>+</sup> binding site, yellow ball stick model; Phe binding site, red ball stick model.

Comparison of structural adaptation characteristics and amino acid substitutions between PsLeuDH and BsLeuDH was shown in Table 1. It can be seen that PsLeuDH exhibited several cold-adapted features. Firstly, the number of electrostatic interactions of PsLeuDH was less than BsLeuDH, which might make the structure of PsLeuDH more flexible [17]. PsLeuDH also had less hydrophobic interactions compared to BsLeuDH, it might make PsLeuDH less rigid and contributed to decrease in structural stability [18]. Secondly, PsLeuDH revealed higher glycine residues and fewer proline and arginine residues that could affect the cold-adapted proteins properties which might offer higher flexibility to proteins [19]. Several amino acid residues in BsLeuDH were replaced by glycine residues in PsLeuDH. The glycine residues might improve the flexibility of the active site, and regulate the entropy of protein unfolding [10], thus probably improving the catalytic efficiency of the enzyme at low temperature. Additionally, proline might reduce the configuration entropy of the unfolding of protein molecules [20] and reduce the stability of enzyme molecules. Additionally, the stability of enzyme was also a significant factor to determine its catalytic characteristics. Some arginine residues in PsLeuDH were replaced by other residues at the same position in BsLeuDH. One of the stability factors in protein structure referred to salt bridges formed by arginine residues [19], arginine might make protein molecules more stable through ionic interaction. Compared with mesophilic enzyme BsLeuDH, PsLeuDH had higher flexibility and lower thermal stability, resulting in higher catalytic efficiency at low temperature [21].


**Table 1.** Comparison of structural adaption features and amino acid substitutions between PsLeuDH and its homolog (BsLeuDH).
