Stereoinversion via Alcohol Dehydrogenases Enables Complete Catabolism of ß-1-Type Lignin-Derived Aromatic Isomers.

Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened ß-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was as yet unknown. Here, we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the Cα hydroxy groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of ß-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four ß-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo-DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCE In many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with ß-1 linkages. Therefore, elucidating the catabolic system of ß-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. Here, we found that threo-DGPD was converted by successive stereoselective oxidation and reduction at the Cα position by multiple alcohol dehydrogenases to erythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native ß-1-type dimer catabolic system.

Hydroxylation with Unusual Stereoinversion Catalyzed by an FeII /2-OG Dependent Oxidase and 3,6-Diene-2,5-diketopiperazine Formation in the Biosynthesis of Brevianamide†K.

Natural products with the 3,6-diene-2,5-diketopiperazine core are widely distributed in nature; however, the biosynthetic mechanism of 3,6-diene-2,5-diketopiperazine in fungi remains to be further elucidated. Through heterologous expression and biochemical investigation of an FeII /2-oxoglutarate-dependent oxidase (AspE) and a heme-dependent P450 enzyme (AspF), we report that AspE, AspF and subsequent dehydration account for the formation of the 3,6-diene-2,5-diketopiperazine substructure of brevianamide K from Aspergillus sp. SK-28, a symbiotic fungus of mangrove plant Kandelia candel. More interestingly, in-depth investigation of the enzymatic mechanism showed that AspE promotes hydroxylation of brevianamide Q with unprecedented stereoinversion through hydrogen atom abstraction and water nucleophilic attack from the opposite face of the resultant iminium cation intermediate.

Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory.

BACKGROUND: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli. RESULTS: To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM L-Phe to D-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids to their corresponding D-amino acids. CONCLUSIONS: The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of D-amino acids via stereoinversion.

Computational Studies on Water-Catalyzed Mechanisms for Stereoinversion of Glutarimide Intermediates Formed from Glutamic Acid Residues in Aqueous Phase.

Aspartic acid (Asp) residues are prone to non-enzymatic stereoinversion, and Asp-residue stereoinversion is believed to be mediated via a succinimide (SI) intermediate. The stereoinverted Asp residues are believed to cause several age-related diseases. However, in peptides and proteins, few studies have reported the stereoinversion of glutamic acid (Glu) residues whose structures are similar to that of Asp. We previously presumed that Glu-residue stereoinversion proceeds via a glutarimide (GI) intermediate and showed that the calculated activation barriers of SI- and GI-intermediate stereoinversion are almost equivalent in the gas phase. In this study, we investigated the stereoinversion pathways of the l-GI intermediate in the aqueous phase using B3LYP density functional methods. The calculated activation barrier of l-GI-intermediate stereoinversion in the aqueous phase was approximately 36 kcal·mol-1, which was much higher than that in the gas phase. Additionally, as this activation barrier exceeded that of Asp-residue stereoinversion, it is presumed that Glu-residue stereoinversion has a lower probability of proceeding under physiological conditions than Asp-residue stereoinversion.

Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues.

Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances <3.0 Šwas greater for exon 26A-2 peptide than for exon 26A-1 peptide. Furthermore, PSA for amide nitrogen of the (n + 1) residue was larger for exon 26A-2 peptide than for exon 26A-1 peptide. These results indicated that the flexibility of Asp and (n + 1) residues and hydrophilicity of peptides, especially in the (n + 1) residue, play important roles in the stereoinversion of Asp. This article is part of a Special Issue entitled: D-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

Catalytic Stereoinversion of L-Alanine to Deuterated D-Alanine.

A combination of an achiral pyridoxal analogue and a chiral base has been developed for catalytic deuteration of L-alanine with inversion of stereochemistry to give deuterated D-alanine under mild conditions (neutral pD and 25 °C) without the use of any protecting groups. This system can also be used for catalytic deuteration of D-alanine with retention of stereochemistry to give deuterated D-alanine. Thus a racemic mixture of alanine can be catalytically deuterated to give an enantiomeric excess of deuterated D-alanine. While catalytic deracemization of alanine is forbidden by the second law of thermodynamics, this system can be used for catalytic deracemization of alanine with deuteration. Such green and biomimetic approach to catalytic stereocontrol provides insights into efficient amino acid transformations.

Highly modular C(1) -symmetric chiral (P,N) ligands with a stereolabile P center: experimental and theoretical studies.

An improved synthesis of a novel class of bidentate (P,N) ligands is presented, the structures of which are characterized by three distinct elements of chirality. The stereoselective installation of the elements of central chirality (at the benzylic carbon and the phosphorus atom) depends on the size of the phosphorus substituent. Thermal inversion of the phosphorus center has been studied experimentally and further correlated by DFT calculations. The potential of these ligands and the role of the phosphorus atom in the asymmetric α-arylation of aldehydes (Pd) and hydrogenation of allylic alcohols (Ir) have also been investigated.

Tertiary alcohols as substrates for S(N)2-like stereoinversion.

Rewrite the textbooks! The stereospecific bimolecular substitution reaction (SN 2) is usually limited to primary and secondary electrophiles. The Shenvi group has developed a method in which tertiary alcohol substrates are converted into isocyanides with configurational inversion. Intriguingly, tertiary hydroxy groups react selectively in the presence of unprotected primary and secondary hydroxy groups.

Highly stereoselective recognition and deracemization of amino acids by supramolecular self-assembly.

The highly stereoselective supramolecular self-assembly of α-amino acids with a chiral aldehyde derived from binol and a chiral guanidine derived from diphenylethylenediamine (dpen) to form the imino acid salt is reported. This system can be used to cleanly convert D-amino acids into L-amino acids or vice versa at ambient temperature. It can also be used to synthesize α-deuterated D- or L-amino acids. A crystal structure of the ternary complex together with DFT computation provided detailed insight into the origin of the stereoselective recognition of amino acids.

Microsolvation and sp(2)-stereoinversion of monomeric α-(2,6-di-tert-butylphenyl)vinyllithium as measured by NMR.

The ß-unsubstituted title compound dissolves in THF as a uniformly trisolvated monomer, whereas it forms exclusively disolvated monomers in tert-butyl methyl ether, Et2O, TMEDA, or toluene with TMEDA (1.4 equiv). This was established at low temperatures through the observation of separated NMR signals for free and lithium-coordinated ligands and/or through the patterns and magnitudes of (13)C,(6)Li NMR coupling constants. An aggregated form was observed only with Et2O (2 equiv) in toluene as the solvent. The olefinic geminal interproton coupling constants of the H2C= part can be used as a secondary criterion to differentiate between these differently solvated ground-states (3, 2, or <2 coordinated ligands per Li). Due to a kinetic trisolvation privilege of THF, the cis/trans sp(2)-stereoinversion rates could be measured through analyses of (1)H NMR line broadening and coalescence only in THF as the solvent: The pseudomonomolecular (because THF-catalyzed), ionic mechanism is initialized by a C-Li bond heterolysis with the transient immobilization of one additional THF ligand, followed by stereoinversion of the quasi-sp(2)-hybridized carbanionic center in cooperation with a "conducted tour" migration of Li(+)(THF)4 along the α-aryl group within the solvent-separated ion pair.

Sterically Unprotected Nucleophilic Boron Cluster Reagents.

A cornerstone of modern synthetic chemistry rests on the ability to manipulate the reactivity of a carbon center by rendering it either electrophilic or nucleophilic. However, accessing a similar reactivity spectrum with boron-based reagents has been significantly more challenging. While classical nucleophilic carbon-based reagents normally do not require steric protection, readily accessible, unprotected boron-based nucleophiles have not yet been realized. Herein, we demonstrate that the bench stable closo-hexaborate cluster anion can engage in a nucleophilic substitution reaction with a wide array of organic and main group electrophiles. The resulting molecules containing B‒C bonds can be further converted to tricoordinate boron species widely used in organic synthesis.

Enantioselective Resolution of (R,S)-Carvedilol to (S)-(-)-Carvedilol by Biocatalysts.

Among the microorganisms employed in the study, Aspergillus niger (GUFCC5443), Escherichia coli (ATCC9637), Streptomyces halstedii (CKM-2), Pseudomonas putida (NCIB9494), Cunninghamella elegans (NCIM689) and Sphingomonas paucimobilis (NCTC11030) were capable for the enantioselective conversion of racemic Carvedilol. Immobilization technique enhanced the enantioselectivity of microorganisms and thus increased the enantiomeric purity of the drug. Excellent enantiomeric ratios (E) were found in reactions catalyzed by immobilized A. niger and E. coli with values 174.44 and 104.26, respectively. Triacylglycerol lipase from Aspergillus niger was also employed in this study as a biocatalyst which resulted in the product with 83.35% enantiomeric excess (ee) and E of 11.34 while the enzyme on immobilization has yielded 99.08% ee and 216.39 E. The conversion yield (C%) of the drug by free-enzyme was 57.42%, which was enhanced by immobilization to 90.51%. Hence, our results suggest that immobilized triacylglycerol lipase from A. niger (Lipase AP6) could be an efficient biocatalyst for the enantioselective resolution of racemic Carvedilol to (S)-(-)-Carvedilol with high enantiomeric purity followed by immobilized cultures of A. niger and E. coli.