# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 1 Introduction  
From the 20 th century onwards, the utilization of enzymes in industrial settings has gained considerable significance, propelled by advancements in technology and scientific breakthroughs, particularly in the field of biochemistry. With enzymes being extensively commercialized and employed, the enzyme market has become more appealing [1]. Proteases (EC 3.4.X.X) catalyze the hydrolysis of peptide bonds within proteins and peptides. Also called proteolytic enzymes or proteinases, they play crucial roles in various metabolic processes and find extensive applications in industries such as detergent manufacturing, pharmaceuticals, and food processing [2]. During environmental stress, particularly under conditions like light or nutrient scarcity, and during cell apoptosis, protease activity intensifies in microalgae metabolism [3]. Proteolysis additionally regulates organelle senescence and facilitates heterocyst formation in cyanobacteria [4]. Proteases are broadly categorized into endopeptidase and exceptidases based on their cleavage site on the substrate. Exceptidases cleave peptide bonds near the amino or carboxy termini, whereas endopeptidases cleave bonds further away from these termini. Further classification is based on the functional group present at their active site, leading to four main groups: (i) serine proteases (EC 3.4.21), (ii) aspartic proteases (EC 3.4.23), (iii) cysteineproteases (also referred to as thiol proteases) (EC 3.4.22), and (iv) metalloproteinases (EC 3.4.17 and EC 3.4.24) [5]. Additionally, there are proteases, such as ATP-dependent proteases, that require ATP for their enzymatic activity, which fall outside these standard classifications [3]. Metal-loproteases are classified further into four groups based on their specificity of action: neutral, alkaline, Myxobacter I and Myxobacter II. Neutral proteases exhibit specificity for hydrophobic amino acids, whereas alkaline proteases have a broader specificity. Myxobacter protease I targets small amino acids (Alanine, Glycine, Cysteine, Serine) surrounding the peptide bond, while specifically cleaving at lysine residues on the amino side of the bond [6]. In this context, the focus is on the crucial role played by the FtsH family of proteases. These enzymes are membrane-anchored, ATP-dependent, zinc metalloproteases (EC 3.4.24) that play a vital role in regulating physiological processes and facilitating adaptation. They are found universally across prokaryotes and in the mitochondria and chloroplasts of eukaryotic cells. FtsH metalloproteases are characterized by N-terminally located transmembrane segments and a central cytosolic region housing AAA-ATPase and Zn\({}^{2+}\) domains. These enzymes fulfill dual roles as proteases and chaperones, crucial for maintaining cellular homeostasis [7]. Their chaperone activities have been extensively studied in bacteria, mitochondria, and chloroplasts [7; 8; 9]. They play a crucial role in regulating the turnover of LpxC deacetylase, which is vital for maintaining the lipopolysaccharide/phospholipid ratio [10]. FtsH proteases are essential for the quality control of membrane proteins by rapidly removing abnormal proteins in prokaryotes, mitochondria, and chloroplasts. Given the susceptibility of the photosynthesis and respiration pathways to oxidative damage, proteins are often short-lived because of damage from reactive oxygen species [11; 12]. FtsH complexes can recognize and remove incorrectly folded proteins [13; 14; 15]. In cyanobacteria belonging to the _Synechocystis_ genus, three classes of FtsH complexes exist in cytoplasmic membranes. The first class, FtsH1/FtsH3, regulates iron acquisition [11; 16; 17], while the second class, FtsH2/FtsH3, is anchored in the thylakoid membranes and is involved in PSII repair, inorganic carbon assimilation, and cosmoregulation [16; 18; 19]. The third class (FtsH4), located especially in thylakoid membranes, has an unclear role [11; 16; 20; 21]. Photosynthetic eukaryotes exhibit greater number of FtsH subunits compared with their ancestors.