Recombinant 2-Deoxyribose-5-phosphate Aldolase in Organic Synthesis: Use of Sequential Two-Substrate and Three-Substrate Aldol Reactions

Wong, Chi‐Huey; García‐Junceda, Eduardo; Chen, Lih-Ren; Blanco, Olga María; Gijsen, Harrie J. M.; Steensma, Darryl H.

doi:10.1021/ja00117a003

Cited by 111 publications

(59 citation statements)

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“…Determination of Enzyme Activity-Assay for DERA activity was conducted at 50°C essentially as described by Wong et al (19). The activity was determined by measuring the oxidation of NADH in a coupled assay with triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase.…”

Section: Methodsmentioning

confidence: 99%

The First Crystal Structure of Archaeal Aldolase

Sakuraba

Tsuge

Shimoya

et al. 2003

Journal of Biological Chemistry

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A gene encoding a 2-deoxy-D-ribose-5-phosphate aldolase (DERA) homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. The enzyme is an extremely thermostable DERA; its activity was not lost after incubation at 100°C for 10 min. The enzyme has a molecular mass of ϳ93 kDa and consists of four subunits with an identical molecular mass of 24 kDa. This is the first report of the presence of tetrameric DERA. The three-dimensional structure of the enzyme was determined by x-ray analysis. The subunit folds into an ␣/␤-barrel. The asymmetric unit consists of two homologous subunits, and a crystallographic 2-fold axis generates the functional tetramer. The main chain coordinate of the monomer of the A. pernix enzyme is quite similar to that of the E. coli enzyme. There was no significant difference in hydrophobic interactions and the number of ion pairs between the monomeric structures of the two enzymes. However, a significant difference in the quaternary structure was observed. The area of the subunit-subunit interface in the dimer of the A. pernix enzyme is much larger compared with the E. coli enzyme. In addition, the A. pernix enzyme is 10 amino acids longer than the E. coli enzyme in the N-terminal region and has an additional N-terminal helix. The N-terminal helix produces a unique dimer-dimer interface. This promotes the formation of a functional tetramer of the A. pernix enzyme and strengthens the hydrophobic intersubunit interactions. These structural features are considered to be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of hyperthermophilic DERA and of aldolase from the Archaea domain.Aldolases catalyze carbon-carbon bond formation and cleavage and are attractive as synthetic catalysts due to their ability to produce stereospecific carbohydrates (1). They are divided into two major classes based on the mode of stabilization of reaction intermediates. Class I and II aldolases employ a Schiff base mechanism (2, 3) and a divalent metal ion for intermediate stabilization (4), respectively. 2-Deoxy-D-ribose-5-phosphate aldolase (DERA 1 ; EC 4.1.2.4) belongs to the class I aldolases and catalyzes a reversible aldol reaction between acetaldehyde and D-glyceraldehyde 3-phosphate to generate 2-deoxy-D-ribose 5-phosphate. DERA is unique in catalyzing the aldol reaction between two aldehydes as both the aldol donor and acceptor components. Its broad substrate specificity is an attractive characteristic for the production of a variety of stereospecific materials (5). The enzyme has high potential utility as an environmentally benign alternative to chiral transition metal catalysis of the asymmetric aldol reaction (6). However, the practical application of DERA is still not successful because of its low stability.Since DERA was first described by Racker (7), who reported the presence of DERA in mammalian tissue as well as in Esc...

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Section: Methodsmentioning

confidence: 99%

The First Crystal Structure of Archaeal Aldolase

Sakuraba

Tsuge

Shimoya

et al. 2003

Journal of Biological Chemistry

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“…However, enzyme-catalyzed aldol condensations can occur at neutral pH and room temperature and display the great potential in the coming future [3]. The class I aldolase, 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4), reversibly catalyzes the aldol reaction of acetaldehyde with D-glyceraldehyde-3-phosphate to form 2-deoxyribose-5-phosphate via a Schiff base intermediate between the active-site lysine and acetaldehyde [4,5]. Unlike other aldolases, both substrates and the product of DERA are aldehydes; therefore, DERA can perform sequential aldol reactions [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

The Fed-Batch Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase (DERA) in Escherichia coli by Exponential Feeding Strategy Control

Pei

Wang

Qiu

et al. 2010

Appl Biochem Biotechnol

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2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (micro(set)) was evaluated at 0.20, 0.15, and 0.10 h(-1), respectively. The DERA concentration and volumetric productivity were associated with micro(set). The cells synthesized the enzyme most efficiently at micro(set) = 0.15 h(-1). The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L(-1) h(-1)) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.

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“…This is because aldolases specific for aldose-type sugars are scarce in nature; 2-deoxyribose-5-phosphate aldolase (DERA) is a notable exception and actually functions as a deoxysugar aldolase. [22][23][24][25][26] Hence, cross-aldol reactions of aldehydes have been a limited field for biocatalysis, and DERA is the only enzyme known to catalyze the stereoselective cross-aldol addition of acetaldehyde to other aldehydes. However, the low conversion rates of this enzyme with non-phosphorylated, unnatural substrates and its inability to generate two consecutive hydroxylated positions with each newly formed bond limit considerably its scope of applicability.…”

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confidence: 99%