D-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, D-amino acids have recently received more and more attention. Enzymes including D-hydantoinase, N-acyl-D-amino acid amidohydrolase, D-amino acid amidase, D-aminopeptidase, D-peptidase, L-amino acid oxidase, D-amino acid aminotransferase, and D-amino acid dehydrogenase can be used for D-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, D-amino acid dehydrogenase method not only produces D-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention.

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 ...

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meso-Diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) is the first member of the meso-DAPDH family known to catalyze the asymmetric reductive amination of 2-keto acids to produce D-amino acids. It is important to understand the catalytic mechanisms of StDAPDH and other enzymes in this family. In this study, based on an evolutionary analysis and examination of catalytic activity, the meso-DAPDH enzymes can be divided into two types. Type I showed highly preferable activity toward meso-diaminopimelate (meso-DAP), and type II exhibited obviously reversible amination activity with a broad substrate spectrum. StDAPDH belongs to type II. A quaternary structure analysis revealed that insertions/deletions (indels) and a loss of quaternary structure resulted in divergence among members of the meso-DAPDH family. A structure alignment of StDAPDH with a representative of type I, the meso-DAPDH from Corynebacterium glutamicum (CgDAPDH), indicated that they had the same folding. Based on sequence and conservation analyses, two amino acid residues of StDAPDH, R35 and R71, were found to be highly conserved within type II while also distinct from each other between the subtypes. Site mutagenesis studies identified R71 as a substrate preference-related residue of StDAPDH, which may serve as an indicator of the amination preference of type II. These results deepen the present understanding of the meso-DAPDH family and provide a solid foundation for the discovery and engineering of meso-DAPDH for D-amino acid biosynthesis.IMPORTANCE The L-form of amino acids is typically more abundant than the D-form. However, the D-form has many important pharmaceutical applications. mesoDiaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH) was the first member of meso-DAPDH known to catalyze the amination of 2-keto acids to produce D-amino acids. Accordingly, we analyzed the evolution of meso-DAPDH proteins and found that they form two groups, i.e., type I proteins, which show high preference toward meso-diaminopimelate (meso-DAP), and type II proteins, which show a broad substrate spectrum. We examined the differences in sequence, ternary structure, and quaternary structure to determine the mechanisms underlying the functional differences between the type I and type II lineages. These results will facilitate the identification of additional meso-DAPDHs and may provide guidance to protein engineering studies for D-amino acid biosynthesis.

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To identify potential therapeutic targets for pulmonary fibrosis induced by silica, we studied the effects of this disease on the expression of microRNAs (miRNAs) in the lung. Rattus norvegicus pulmonary silicosis models were used in conjunction with high-throughput screening of lung specimens to compare the expression of miRNAs in control and pulmonary silicosis tissues. A total of 70 miRNAs were found to be differentially expressed between control and pulmonary silicosis tissues. This included 41 miRNAs that were upregulated and 29 that were downregulated relative to controls. Among them, miR-292-5p, miR-155-3p, miR-1193-3p, miR-411-3p, miR-370-3p, and miR-409a-5p were found to be similarly altered in rat lung and transforming growth factor (TGF)-b1-induced cultured fibroblasts. Using miRNA mimics and inhibitors, we found that miR-1193-3p, miR-411-3p, and miR-370-3p exhibited potent anti-fibrotic effects, while miR-292-5p demonstrated profibrotic effects in TGF-b1-stimulated lung fibroblasts. Moreover, we also found that miR-411-3p effectively reduced pulmonary silicosis in the mouse lung by regulating Mrtfa expression, as demonstrated using biochemical and histological assays. In conclusion, our findings indicate that miRNA expression is perturbed in pulmonary silicosis and suggest that therapeutic interventions targeting specific miRNAs might be effective in the treatment of this occupational disease.

In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ؎ 0.06 U · mg ؊1 , which was 35.1-fold enhancement over the wild-type enzyme. D-Amino acids have attracted great attention because of their increasing importance as chiral building blocks for the synthesis of pharmaceuticals and food ingredients (1-3). Peptides containing D-amino acids show high resistance against proteolytic degradation, which is an important factor in drug design (4). D-pHydroxyphenylglycine has been widely used as precursor for the synthesis of some antibiotics and semisynthetic antibiotics, such as amoxicillin, ampicillin, and cefbuparzone (5, 6). D-Phenylalanine is the important chiral component of nateglinide, a drug for the treatment of type 2 diabetes (7). The dipeptide artificial sweeter alitame contains a D-alanine moiety (8). Among the biocatalytic methods for the preparation of D-amino acids, a useful and straightforward approach is the reductive amination of 2-keto acids catalyzed by D-amino acid dehydrogenases (EC1.4.99.1; D-AADH) (9, 10). Unfortunately, the characteristics of membrane-bound native D-AADH have hindered the industrial application of this family of enzymes (11). meso-Diaminopimelate dehydrogenase (EC1.4.1.16; meso-DAPDH) is a special class of D-AADHs which catalyze the reversible oxidative deamination and reductive amination at the D-center of meso-2,6-diaminopimelate (meso-DAP) with high stereoselectivity (12), restricting its application in D-amino acid synthesis. In this context, VedhaPeters et al. expanded the substrate profile of the meso-DAPDH from Corynebacterium glutamicum by combining site saturation and random mutagenesis, resulting in a few mutants with activity toward a series of 2-keto acids (11). 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.The three-dimensional structure of the meso-DAPDH from C. glutamicum in a ternary complex showed that the substrate/inhibitor binding residues were Asn253, Gln150, Gly151, Asp90, and Asp120 for the D-center of meso-DAP and His244, Thr171, Arg195, Trp144 for its L-center (14). It was reported that the C ␣ -hydrogen of the D-center was transferred to the C-4= of the nicotinamide ring to form an imine intermediate during deamination, and the distal L-center only maintained the correct orientation of meso-DAP (15,16). In order to enlarge the substrate binding pocket without ch...

The conjugation protocols in myxobacterium Sorangium cellulosum are often inapplicable due to the strain-specific sensitivity to the presence of Escherichia coli cells or the resistances to many antibiotics. Here we report that the conjugative transfer of the mobilizable plasmid pCVD442 from E. coli DH5alpha (lambda pir) to Sorangium strains could be greatly increased by the presence of low doses of dual selection antibiotics in the mating medium. The improvement was efficient in either E. coli-tolerant or sensitive Sorangium strains. For those phleomycin and hygromycin tolerant Sorangium strains, chloramphenicol-resistance gene was developed as a new selectable marker by driving the resistance gene with the aphII promoter. Using the improved protocol, the epothilone biosynthetic pathway was inactivated by an insertion mutation in the biosynthetic genes of the producing Sorangium strains.