Aromatic l ‐amino acid decarboxylases (AADCs) catalyze the release of CO2 from proteinogenic and non‐proteinogenic l ‐amino acid substrates and are involved in pathways that biosynthesize neurotransmitters or bioactive natural products. In contrast to AADCs from animals and plants, fungal AADCs have received very little attention. Here, we report on the in vitro characterization of heterologously produced Ceriporiopsis subvermispora AADC, now referred to as CsTDC, which is the first characterized basidiomycete AADC. This study identified the enzyme as a decarboxylase that is strictly specific for l ‐tryptophan and 5‐hydroxy‐l ‐tryptophan. The tdc gene was subjected to saturation mutagenesis so as to vary the key active site residue, Gly351. Aliphatic amino acid residues, l ‐serine, or l ‐threonine at position 351 added l ‐tyrosine and 3,4‐dihydroxy‐l ‐phenylalanine (l ‐DOPA) decarboxylase activity while retaining stereospecificity and l ‐tryptophan decarboxylase activity. 相似文献
β‐Methyltryptophans (β‐mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic‐based therapeutics. Currently β‐mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l ‐threonine and various indoles to generate β‐mTrp and derivatives in a single step. Although l ‐serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l ‐Thr as a substrate. The condensation of l ‐Thr and indole proceeds with retention of configuration at both α‐ and β‐positions to give (2S,3S)‐β‐mTrp. The integration of StTrpS (βL166V) with l ‐amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d ‐configured and regioselectively halogenated or arylated β‐mTrp derivatives. 相似文献
Proline hydroxylases are iron(II)/2‐oxoglutarate‐dependent enzymes that hydroxylate l ‐proline and derivatives, such as l pipecolic acid, which is the six‐membered‐ring homologue of l ‐proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l ‐pipecolic acid and trans‐3‐ and trans‐4‐methyl‐l ‐proline, but virtually no l ‐proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l ‐proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l ‐proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site‐directed mutagenesis in GetF did not yield variants that accepted l ‐proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l ‐proline relative to that of preferred substrates. 相似文献
The 2‐O‐α‐d ‐glucoside of l ‐ascorbic acid (AA‐2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA‐2G is currently produced through biocatalytic glucosylation of l ‐ascorbic acid from starch‐derived oligosaccharides. Sucrose would be an ideal substrate for AA‐2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8–6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2‐O‐α‐glucosylation of l ‐ascorbic acid from sucrose with high efficiency and perfect site‐selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L?1; 460 mm ), from which pure AA‐2G was readily recovered in ~50 % overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a “reverse‐protonation” mechanism in which the catalytic base Glu232 on the glucosyl–enzyme intermediate must be protonated for attack on the anomeric carbon from the 2‐hydroxyl of the ionized l ‐ascorbate substrate. 相似文献
Due to their diverse regio‐ and stereoselectivities, proline hydroxylases provide a straightforward access to hydroxprolines and other hydroxylated cylic amino acids, valuable chiral building blocks for chemical synthesis, which are often not available at reasonable expense by classical chemical synthesis. As yet, the application of proline hydroxylases is limited to a sophisticated industrial process for the production of two hydroxyproline isomers. This is mainly due to difficulties in their heterologues expression, their limited in vitro stability and complex product purification procedures. Here we describe a facile method for the production of cis‐3‐, cis‐4‐ and trans‐4‐proline hydroxylase, and their application for the regio‐ and stereoselective hydroxylation of L ‐proline and its six‐membered ring homologue l‐ pipecolic acid. Since in vitro catalysis with these enzymes is not very efficient and conversions are restricted to the milligram scale, an in vivo procedure was established, which allowed a quantitative conversion of 6 mM l‐ proline in shake flask cultures. After facile product purification via ion exchange chromatography, hydroxyprolines were isolated in yields of 35–61% (175–305 mg per flask). L ‐Pipecolic acid was converted with the isolated enzymes to prove the selectivities of the reactions. In transformations with optimized iron(II) concentration, conversions of 17–68% to hydroxylated products were achieved. The regio‐ and stereochemistry of the products was determined by NMR techniques. To demonstrate the applicability of the preparative in vivo approach for non‐physiological substrates, L ‐pipecolic acid was converted with an E. coli strain producing trans‐4‐proline hydroxylase to trans‐5‐hydroxy‐L ‐pipecolic acid in 61% yield. Thus, a synthetically valuable group of biocatalysts was made readily accessible for application in the laboratory without a need for special equipment or considerable development effort. 相似文献
A one‐pot, two‐step biocatalytic platform for the regiospecfic C‐methylation and C‐ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S‐adenosyl‐l ‐methionine (SAM), followed by alkylation of aromatic substrates by the C‐methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled 13CH3,13CD3 and CD3 groups from their corresponding SAM analogues formed in situ. 相似文献
Non‐cationic and amphipathic indoloazepinone‐constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l ‐Aia‐Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc‐[l ‐Aia‐Gly]2,4‐OBn oligomers 12 and 13 and Boc‐[l ‐Aia‐β3‐h‐l ‐Ala]2,4‐OBn oligomers 16 and 17 were totally or partially disordered, Boc‐[l ‐Aia‐l ‐Ala]2‐OBn ( 14 ) induced a typical turn stabilized by C5‐ and C7‐membered H‐bond pseudo‐cycles and aromatic interactions. Boc‐[l ‐Aia‐l ‐Ala]4‐OBn ( 15 ) exhibited a unique structure with remarkable T‐shaped π‐stacking interactions involving the indole rings of the four l ‐Aia residues forming a dense hydrophobic cluster. All of the proposed FITC‐6‐Ahx‐[l ‐Aia‐Xxx]4‐NH2 oligomers 19 – 23 , with the exception of FITC‐6‐Ahx‐[l ‐Aia‐Gly]4‐NH2 ( 18 ), were internalized by MDA‐MB‐231 cells with higher efficiency than the positive references penetratin and Arg8. In parallel, the compounds of this series were successfully explored in an in vitro blood–brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac‐[l ‐Aia‐Xxx]4‐NH2 oligomers in the PAMPA model, Ac‐[l ‐Aia‐l ‐Arg]4‐NH2 ( 26 ) showed significant permeation in the in vitro cell‐based human model of the BBB, suggesting an active mechanism of cell penetration. 相似文献
Although bacterial iterative type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products is poorly understood. We report the functional characterization of the putative orsellinic acid C2‐O‐methyltransferase, which is involved in calicheamicin biosynthesis. This study suggests that C2‐O‐methylation precedes C3‐hydroxylation/methylation and C5‐iodination and requires a coenzyme A‐ or acyl carrier protein‐bound substrate. 相似文献
MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271. 相似文献
An efficient multi‐enzyme cascade reaction for the synthesis of (R)‐ or (S)‐2‐hydroxybutyric acid [(R)‐ or (S)‐2‐HB] from l ‐threonine was developed by using recombinant Escherichia coli cells expressing separately or co‐expressing l ‐threonine deaminase from Escherichia coli K‐12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l ‐lactate dehydrogenase (l ‐LDH) from Oryctolagus cuniculus or d ‐lactate dehydrogenase (d ‐LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l ‐threonine were completely transformed to (R)‐ or (S)‐2‐HB in optically pure form (>99% ee) with high isolated yields. This one‐pot multi‐enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds.
Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure. 相似文献
Capuramycins are one of several known classes of natural products that contain an l ‐Lys‐derived l ‐α‐amino‐?‐caprolactam (l ‐ACL) unit. The α‐amino group of l ‐ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP‐independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP‐dependent amide‐bond‐forming catalyst responsible for the biosynthesis of the remaining amide bond present in l ‐ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l ‐Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS‐mediated mechanism is employed in the biosynthesis of other l ‐ACL‐containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS‐derived peptides that contain an unmodified l ‐Lys residue. 相似文献
We report a β‐hairpin dual stabilizing strategy: a d ‐proline‐l ‐proline (d ‐Pro‐l ‐Pro) dipeptide as the nucleating turn, and a thioether tether as a side‐chain linkage at a precisely designed position to stabilize the β‐hairpin. This method was used to modify the C‐terminal β‐hairpin moiety of the plant defensin, pv‐defensin, in order to obtain a stabilized peptide with enhanced anti‐Candida albicans activity (MIC 84–3.0 μm ), high serum stability (50 % remaining after 48 h) and low hemolysis (<10 % at 152 μm ). This modified peptide penetrated the C. albicans cell membrane within 5 min and showed high activity against clinically isolated antibiotic‐resistant C. albicans and Candida glabrata strains. 相似文献