Probing the Essential Catalytic Residues and Substrate Affinity in the Thermoactive Bacillus stearothermophilus US100 l -Arabinose Isomerase by Site-Directed Mutagenesis

Abstract

The L-arabinose isomerase (L-AI) from Bacillus stearothermophilus US100 is characterized by its high thermoactivity and catalytic efficiency. Furthermore, as opposed to the majority of L-arabinose isomerases, this enzyme requires metallic ions for its thermostability rather than for its activity. These features make US100 L-AI attractive as a template for industrial use. Based on previously solved crystal structures and sequence alignments, we identified amino acids that are putatively important for the US100 L-AI isomerization reaction. Among these, E306, E331, H348, and H447, which correspond to the suggested essential catalytic amino acids of the L-fucose isomerase and the L-arabinose isomerase from Escherichia coli, are presumed to be the active-site residues of US100 L-AI. Site-directed mutagenesis confirmed that the mutation of these residues resulted in totally inactive proteins, thus demonstrating their critical role in the enzyme activity. A homology model of US100 L-AI was constructed, and its analysis highlighted another set of residues which may be crucial for the recognition and processing of substrates; hence, these residues were subjected to mutagenesis studies. The replacement of the D308, F329, E351, and H446 amino acids with alanine seriously affected the enzyme activities, and suggestions about the roles of these residues in the catalytic mechanism are given. The mutation F279Q strongly increased the enzyme's affinity for L-fucose and decreased the affinity for L-arabinose compared to that of the wild-type enzyme, showing the implication of this amino acid in substrate recognition.L-Arabinose isomerases (L-AIs; EC 5.3.1.4) catalyze the conversion of L-arabinose to L-ribulose in biological systems. They are also referred to as D-galactose isomerases due to their ability, in vitro, to isomerize D-galactose into D-tagatose (4). The latter sugar is of importance when one considers the privileged position of this isomer of D-galactose in sweeteners. D-Tagatose is a rare natural ketohexose having a taste and physical properties similar to those of sucrose (24). Additionally, D-tagatose is an antihyperglycemic factor, an efficient antibiofilm with a very low-calorie carbohydrate, and a bulking agent (20,36). It has been the subject of recent interest in the food and drug industry and is considered to be a safe and low-calorie substance in the United States (17).The majority of L-AIs previously described, such as those from Escherichia coli and Mycobacterium and Lactobacillus species, are not thermoactive (9, 35). Isomerization at high temperatures increases the reaction rate and allows a shift in the equilibrium between D-galactose and D-tagatose towards the latter, which is desirable for industrial use (12). For this reason, many thermoactive and thermostable L-AIs have been isolated and studied, including those derived from members of the genera Thermotoga, Geobacillus, Thermoanaerobacter, and Thermus (10,14,17,18).In their functional conformations, the L-AIs have been shown to adopt a hexameric q...

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

“…The multiple sequence alignment of Arthrobacter sp. 22c L-arabinose isomerase (22cAI) with its previously described counterparts from psychrotolerant, mesophilic, thermophilic and hyperhtermophilic microorganisms revealed some conserved regions and four essential catalytic residues, namely E307, E334, H351 and H451, corresponding to the E306, E333, H350 and H450 of E. coli L-arabinose isomerase and the E306, E331, H348 and H447 of B. stearothermophilus US100 L-AI (Figure 1) [35,36]. …”

Thermostable L‐arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D‐tagatose production: gene cloning, purification and characterisation

“…The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly and active site architecture strongly suggests that the two enzymes have functional similarities. Both ECFI and ECAI convert aldoses into corresponding ketoses using Mn 2+ as a cofactor 14, 24. In the case of BSAI the activity and thermostability increased greatly after the addition of Mn 2+ .…”

“…This bifunctional activity could be exploited industrially for the production of D ‐tagatose. L‐AI has been identified in various micro‐organisms such as Aerobacter aerogenes ,9 Bacillus halodurans ,10 Escherichia coli ,11 Lactobacillus plantarum ,12 Bacillus stearothermophilus ,13, 14 Thermus sp.,15 Thermoanaerobacter mathranii ,16 Thermotoga neapolitana 17 and Thermotoga maritima 18…”

Probing the Molecular Determinant for the Catalytic Efficiency of l -Arabinose Isomerase from Bacillus licheniformis

“…Upon confirming the role of the 4 putative catalytic residues, we turned our focus to 12 other conserved residues within 5 Å of the SBP of B. licheniformis L-AI. The roles of 5 of these 12 conserved residues, F279, D308, F329, E351, and H446, have been described previously (19). The roles of the remaining seven residues (M185, T276, Y333, L345, M349, I370, and W439) were therefore investigated by further site-directed mutagenesis (Fig.…”