Asymmetric adsorption by quartz: a model for the prebiotic origin of optical activity.

One mechanism previously proposed for the abiotic accumulation of molecules of one chirality in nature is asymmetric adsorption on the chiral surfaces of optically active quartz crystals. Earlier literature in this field is reviewed, with the conclusion that previous investigations of this phenomenon, using optical rotation criteria, have afforded ambiguous results. We now have studied the adsorption of radioactive D- and L-alanine on powdered d- and l-quartz, using change in radioactivity level as a criterion for both gross and differential adsorption. d-Quartz preferentially adsorbed D-alanine from anhydrous dimethyl-formamide solution, and l-quartz L-alanine. The differential adsorption varied between 1.0 and 1.8%. The implications of these observations are discussed from the viewpoint of early chemical evolution and the origin of optically active organic compounds in nature.     

Stereoselective hydrolysis of optically active isomeric isopulegyl acetates by rhodotorula mucilaginosa and bacillus sp.

A method for isolation of optically pure l-isopulegol from a mixture of its optically active isomers using the microorganisms Rhodotorula mucilaginosa and Bacillus sp. is described. Microorganisms hydrolyzed l-isopulegyl acetate (26–40%) and in a small degree d-isopulegol acetate whereas the d-neoisopulegol acetate remained non-hydrolyzed. The optical purity of the chromatographie pure l-isopulegol was 97.6%.     

Esterolytic Activity of Acid Carboxypeptidase from Aspergillus saitoi

Asymmetric reduction of aromatic ketones using chirally modified reagents prepared from sodium borohydride and optically active acids in the presence or absence of 1,2: 5,6-di-O-isopropylidene-α-d-glucofuranose produced the corresponding optically active alcohols with optical yields of 4 ~ 47%. The reagent prepared from sodium borohydride and 1 equivalent of l-malic acid in the presence of 2 equivalents of 1,2: 5,6-di-O-isopropylidene-α-d-gluco-furanose gave the highest yields.     

Tagatose: properties, applications, and biotechnological processes

d-Tagatose has attracted a great deal of attention in recent years due to its health benefits and similar properties to sucrose. d-Tagatose can be used as a low-calorie sweetener, as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Biotransformation of d-tagatose has been produced using several biocatalyst sources. Among the biocatalysts, l-arabinose isomerase has been mostly applied for d-tagatose production because of the industrial feasibility for the use of d-galactose as a substrate. In this article, the characterization of many l-arabinose isomerases and their d-tagatose production is compared. Protein engineering and immobilization of the enzyme for increasing the conversion rate of d-galactose to d-tagatose are also reviewed.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

Microwave-assisted synthesis and characterization of optically active poly (ester-imide)s incorporating l-alanine

Pyromellitic dianhydride (1) was reacted with L-alanine (2) to result [N,N′-(pyromellitoyl)-bis-l-alanine diacid] (3). This compound (3) was converted to N,N′-(pyromellitoyl)-bis-l-alanine diacyl chloride (4) by reaction with thionyl chloride. The microwave-assisted polycondensation of this diacyl chloride (4) with polyethyleneglycol-diol (PEG-200) and/or three synthetic aromatic diols furnish a series of new PEIs and Co-PEIs in a laboratory microwave oven (Milestone). The resulting polymers and copolymers have inherent viscosities in the range of 0.31–0.53 dl g⁻¹. These polymers are optically active, thermally stable and soluble in polar aprotic solvents such as DMF, DMSO, NMP, DMAc, and sulfuric acid. All of the above polymers were fully characterized by IR spectroscopy, ¹H NMR spectroscopy, elemental analyses, specific rotation and thermal analyses. Some structural characterizations and physical properties of these optically active PEIs and Co-PEIs have been reported.     

Biomimetic asymmetric oxidative coupling of phenols

This paper describes the first examples of asymmetric induction in the oxidative coupling of phenols using chiral oxidants. When chiral cupric-amine complexes were used as oxidants, low asymmetric induction was achieved in the coupling of naphthols. The formation of optically active d-dehydrogriseofulvin and l-Licarin A using the cupric-l-a-phenylethylamine complex perhaps mimics the action of copper-containing enzymes known to catalyze phenol coupling.     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(?)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(?)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver?CBurk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical ??/??-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 ??-helices and 8 ??-strands, and 2 extra short ??-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Chemical and enzymatic approaches to the synthesis of optically pure ethyl (R)-4-cyano-3-hydroxybutanoate

Ethyl (R)-4-cyano-3-hydroxybutanoate (HN) is an important chiral synthon for side chain of the cholesterol-lowering drug atorvastatin (Lipitor), which is the hydroxymethylglutaryl CoA reductase inhibitor. HN is also used as a synthon in the production of l-carnitine and (R)-4-amino-3-hydroxybutanoic acid. It is necessary to have a clear understanding of the synthesis process of HN for its extensive use. This review gives an overview of different synthetic strategies of optically active HN, including chemical and enzymatic approaches. The emphasis is focused mainly on the synthetic routes using biocatalysts, such as halohydrin dehalogenase, nitrilase, carbonyl reductase, and lipase.     

Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

A Novel Bacterial Enzyme with d-Glucuronyl C5-epimerase Activity

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Inhibition of thermolysin by 3-trimethylsilylalanine derivatives

The inhibition of thermolysin by an optically active silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), and its derivatives was examined by considering the similarity of structure between TMS-Ala and leucine. Although free l- and d-TMS-Ala did not show the inhibition, several derivatives of l-TMS-Ala, especially Z-l-TMS-Ala and l-Leu-(l-TMS-Ala), exhibited a higher inhibitory activity toward thermolysin than did Z-l-Leu and l-Leu-l-Leu respectively. Effects of TMS-Ala on the activity of its derivatives and the mode of interaction between the derivatives of TMS-Ala and thermolysin are also discussed. Received: 24 February 1999 / Received last revision: 4 June 1999 / Accepted: 27 June 1999     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Phase Equilibria in DOPC/DPPC-d62/Cholesterol Mixtures

There is broad interest in the question of fluid-fluid phase coexistence in membranes, in particular, whether evidence for liquid-disordered (l_d)-liquid-ordered (l_o) two-phase regions or membrane “rafts” can be found in natural membranes. In model membrane systems, such phase behavior is observed, and we have used deuterium nuclear magnetic resonance spectroscopy to map the phase boundaries of ternary mixtures containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), chain-perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC-d₆₂), and cholesterol. For both this ternary model system and the binary DPPC-d₆₂/cholesterol sytem, we present clear evidence for l_d-l_o two-phase coexistence. We have selected sample compositions to focus on this region of fluid-fluid phase coexistence and to determine its temperature and composition ranges. The deuterium nuclear magnetic resonance spectra for compositions near the l_d-l_o phase boundary at high cholesterol concentrations show evidence of exchange broadening or critical fluctuations in composition, similar to that reported by Vist and Davis. There appears to be a line of critical compositions ranging from 48°C for a DOPC/DPPC-d₆₂/cholesterol composition of 0:75:25, to ∼−8°C for the composition 57:14:29. At temperatures below this two-phase region, there is a region of three-phase coexistence (l_d-l_o-gel). These results are collected and presented in terms of a partial ternary phase diagram that is consistent with previously reported results of Vist and Davis.     

Protective Effect of dl-Threonine on Bacterial Cells during Freeeze-Drying and Ineffectiveness of its Optically Active Forms

dl-Threonine and dl-allothreonine showed a protective effect on various bacterial cells in the process of freeze-drying whereas l- and d-forms of them did not, probably owing to the difference in the physicochemical characteristics between l- (or d-) form and dl-form of the compounds in question. There was no difference in the protective activity between the optically active and inactive forms in the cases of serine, proline, tartaric acid and pyrrolidonecaboxylic acid.