DERA

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Introduction

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the reversible aldol reaction of acetaldehyde and glyceraldehyde-3-phosphate (G3P) to form 2-deoxyribose-5-phosphate (DR5P). This enzyme is special among aldolases, which use aldehyde instead of ketone as the natural donor. All known DERAs work according to the class I mechanism. DERA can accept many aldehydes with long chains up to four carbon atoms and generate (S)-configured stereogenic center in G3P. Another interesting feature of DERA is that it can catalyze the sequential aldol reaction to produce 2,4,6-trideoxyhexoses, which can be used as a valuable intermediate for atorvastatin and cholesterol-lowering drugs. It is found that the sequential addition of two molecules of acetaldehyde and chloroacetaldehyde can result in a highly stereospecific polyol system, in which the stereo selectivity is controlled by the enzyme rather than the substrate. This sequential aldol reaction is thermodynamically controlled and stops when a stable intramolecular hemiacetal is formed. 

Figure 1. The in vivo 2-deoxy-D-ribose-5-phosphate reaction catalyzed by DERA (Haridas, M.; et al. 2018)

DERA is encoded by the deoC gene. The sequence of DERA_EC was first reported in 1982 when the enzyme was isolated from E. coli strain K-12. The enzyme consists of 259 amino acids and has a molecular weight of 27.7 kDa. The amino acid composition of the enzyme isolated from S. typhimurium has long been reported. In recent years, DERA has been identified in a variety of plant and animal tissues, and these tissues have been characterized according to several parameters. In humans, it is most expressed in lung and liver cells, and participates in the stress response by delaying or minimizing the stress-induced damage in these cells.

The X-ray structure of DERA_EC shows that the enzyme is a common TIM (α/β) ₈-barrel fold. According to the structure analysis, the enzyme exists as a dimer. After comparing the crystal structures of DERA from different organisms, it was found that water molecules are conserved in all crystal structures, indicating its importance in the catalytic mechanism. The structural study of the enzyme isolated from the archaeon Aeropyrum pernix shows that its monomer structure is very similar to that of DERA_EC, but it exists in a stable tetramer structure. The tetramer form plays an important role in improving the thermal stability of the protein, but has no effect on the catalytic activity.

Later crystallographic studies identified a carbinolamine intermediate and a second lysine residue at position 201 (Lys), which is very close to Lys167. Subsequent studies have shown that Lys167 and Lys201 are critical to the catalytic activity of DERA, Lys167 is directly involved in the formation of Schiff base, and Lys201 may be involved in the perturbation of pKa for Lys167.

Figure 2. Two-dimensional diagram shows the active side contacts (Haridas, M.; et al. 2018)

Acetaldehyde resistance of DERA

Jennerwein et al. used high-throughput screening combined with directed evolution strategies to identify mutations that have a positive effect on the catalytic activity of DERA and the chloroacetaldehyde resistance. They found that the hydrophobic clusters formed by amino acid residues Phe200, Ile166 and Met185 are very close to the active sites Lys167 and Lys201 of DERA. On combining the successful point mutations in DERA_EC, they obtained a ten-fold improved variant of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside under industrially relevant conditions.

Scientists often use virtual mutation and site-directed mutagenesis to affect the rigidity of protein structure. Variant mutated at positions 120, 174, and 213 also show enhanced tolerance, but the initial activity is reduced by 50%. Dick et al. identified the aldol condensation product between two acetaldehyde molecules as DERA inhibitors. It is almost impossible to exchange the catalytic residue Lys167 without losing the function of the enzyme. Therefore, the substituting of cysteine with a non-nucleophilic amino acid as methionine is considered to be the best choice for the production of an acetaldehyde resistant enzyme. The Cys47Met variant remains the most aldehyde-resistant DERA_EC variant reported to date.

Figure 3. Crystal structure E. coli DERA (Haridas, M.; et al. 2018)