A Novel meso -Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d -Amino Acid Synthesis

Abstract

meso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP؉ -dependent enzyme which catalyzes the reversible oxidative deamination on the D-configuration of meso-2,6-diaminopimelate to produce L-2-amino-6-oxopimelate. In this study, the gene encoding a meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum was cloned and expressed in Escherichia coli. In addition to the native substrate meso-2,6-diaminopimelate, the purified enzyme also showed activity toward Dalanine, D-valine, and D-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generate D-amino acids in up to 99% conversion and 99% enantiomeric excess. Since meso-diaminopimelate dehydrogenases are known to be specific to meso-2,6-diaminopimelate, this is a unique wild-type meso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential for D-amino acid synthesis. The enzyme is the most stable meso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites of S. thermophilum meso-DAPDH different from the sequences of other known meso-DAPDHs were replaced with the conserved amino acids in other meso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile of S. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effective D-amino acid dehydrogenases by protein engineering. N ot only do D-amino acids serve as specialized components of many types of machineries in living organisms, such as neural signaling (20), and bacterial cell walls (19), but they also are important components or building blocks in the production of pharmaceuticals and other fine chemicals (4, 5, 12). As such, many methods for the synthesis of D-amino acids and their derivatives have been developed. Just as L-amino acid dehydrogenases are useful for preparation of L-amino acids from the corresponding 2-keto acids (6,7,13,21), the use of D-amino acid dehydrogenase (D-AADH) should offer a straightforward approach, in which the enzyme catalyzes the reductive amination of 2-keto acid to give D-amino acid. However, NAD(P)H-dependent D-amino acid dehydrogenase is much less abundant in nature than its L-amino acid counterpart and largely unexplored. The most-known D-AADH is meso-diaminopimelate dehydrogenase (meso-DAPDH; EC 1.4.1.16), which is a key enzyme in the lysine biosynthetic pathway and has been found in bacteria, such as Bacillus sphaericus (17) and Corynebacterium glutamicum (14). In addition, meso-DAPDH has also been isolated from plants, for example, soybeans (Glycine max) (24). meso-DAPDH is NADP ϩ dependent and catalyzes the reversible oxidative deamination on the D-configuration center of meso-2,6-diaminopimelate (meso-DAP) to yield L-2-amino-6-oxopimelate (17). However, previously reported meso-DAPDHs are generally specific toward meso-DAP and showed only very ...

Engineering the meso -Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum by Site Saturation Mutagenesis for d -Phenylalanine Synthesis

“…The mutant enzymes were soluble and could be purified with yields similar to the yield of the wild-type enzyme. The specific activities for the reductive amination of phenylpyruvic acid were measured as described before (13). As shown in Table 1, residue His227 exerted a greater effect on the enzyme activity than residues Thr171 and Arg181.…”

“…Cells were harvested by centrifugation and disrupted with a low-temperature ultrahigh-pressure continuous flow cell disrupter (JNBIO, China). Wild-type and all mutant enzymes were purified on an ÄKTA Avant 25 system (GE Healthcare, USA) with a His trap high-performance (HP) column (GE Healthcare, USA) as described in our previous work (13). The mutant enzymes were soluble and could be purified with yields similar to the yield of the wild-type enzyme.…”

“…In our recent work, the NADP ϩ -dependent meso-DAPDH from Symbiobacterium thermophilum was found to possess relaxed substrate specificity and catalyze the reductive amination of pyruvic acid, yielding D-alanine with 68% yield and 99% enantiomeric excess (ee). Although this enzyme is less active with other bulky 2-keto acids (e.g., phenylpyruvic acid) (13), it should serve as an excellent starting enzyme to be engineered for expanded substrate specificity.…”

“…After being replicated into a new deep 96-well plate, the colonies were incubated at 37°C in a shaker for microtiter plates (INFORS, Switzerland) at 400 rpm for 6 h. After isopropyl-␤-D-thiogalactopyranoside (IPTG; 0.1 mM) was added, the plate was incubated at 30°C for 16 h. Cell pellets were harvested by centrifugation, suspended into 1.5 ml of phosphate buffer (100 mM, pH 7.0), and then disrupted by using an ultrasonic liquid processor (Q700; Qsonica, USA) with 96-tip horns. The resulting supernatants were used for activity screening by monitoring the NADPH consumption at room temperature in a reac-tion system of phosphate buffer (100 mM, pH 7.0) containing 240 l of crude enzyme extract, 50 l of substrate mixture (20 mM phenylpyruvic acid and 200 mM NH 4 Cl), and 10 l of NADPH (0.26 mM) (13). Variants with 2-fold higher activity toward phenylpyruvic acid than wild-type enzyme were selected.…”

“…The site saturation mutagenesis libraries were created individually for each amino acid residue by using a QuikChange mutagenesis protocol (Stratagene) with NNK as a degenerate codon (17). The recombinant plasmid pET32a (ϩ) with the S. thermophilum meso-DAPDH gene reported previously (13) was used as the template with the primers listed in Table S1 in the supplemental material. The PCR product was transformed into BL21(DE3) competent cells for expression.…”