Proline‐specific endoproteases have been successfully used in, for example, the in‐situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the ...generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline‐specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline‐specific endoprotease activity. Family S28 proteases have a conventional Ser‐Asp‐His catalytic triad, but their oxyanion‐stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics‐assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28‐proline‐specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino‐ and carboxyl‐terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.
The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics ...producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam–negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6- epi -pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450 Pᵣₐᵥₐ bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450 Pᵣₐᵥₐ fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.
Significance Statins are successful widely used drugs that decrease the risk of coronary heart disease and strokes by lowering cholesterol levels. They selectively inhibit the key regulatory enzyme of the cholesterol synthesis pathway, thus lowering levels of plasma LDL (bad) cholesterol. Pravastatin is one of the leading and most effective statins, derived from the natural product compactin. However, pravastatin production involves a costly dual-step fermentation and biotransformation process. Here we present a single-step fermentative method for production of the active drug pravastatin. Reprogramming of the antibiotics-producing fungus Penicillium chrysogenum , with discovery and engineering of an enzyme involved in the hydroxylation of compactin, enables high level fermentation of the correct form of pravastatin to facilitate efficient industrial-scale statin drug production.
We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or ...(S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90 % ee and to (S)-epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites.
Penicillium chrysogenum Acyl coenzyme A:isopenicillin N acyltransferase (AT) performs the last step in the biosynthesis of hydrophobic penicillins, exchanging the hydrophilic side chain of a ...precursor for various hydrophobic side chains. Like other N-terminal nucleophile hydrolases AT is produced as an inactive precursor that matures upon posttranslational cleavage. The structure of a Cys103Ala precursor mutant shows that maturation is autoproteolytic, initiated by Cys103 cleaving its preceding peptide bond. The crystal structure of the mature enzyme shows that after autoproteolysis residues 92–102 fold outwards, exposing a buried pocket. This pocket is structurally and chemically flexible and can accommodate substrates of different size and polarity. Modeling of a substrate-bound state indicates the residues important for catalysis. Comparison of the proposed autoproteolytic and substrate hydrolysis mechanisms shows that in both events the same catalytic residues are used, but that they perform different roles in catalysis.
► Maturation of AT (an isopenicillin N converting enzyme) is an intramolecular event ► Substrate conversion and maturation use the same residues with different functions ► The broad substrate specificity of AT is explained by its extensive substrate pocket
Protein redesign by learning from data van den Berg, Bastiaan A; Reinders, Marcel J.T; van der Laan, Jan-Metske ...
Protein engineering, design and selection,
09/2014, Volume:
27, Issue:
9
Journal Article
Peer reviewed
Open access
Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying ...biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5–45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.
We here report four biocatalytic approaches for the synthesis of the protected amino acid building block α-benzyl
L
-glutamate. Screenings of these routes to identify active and selective enzymes ...were conducted, and major hits were confirmed in retest reactions. In the first approach,
N
-Boc
L
-glutamic acid is mono-benzylesterified by the protease Alcalase with 81% yield; and in the other three approaches, a biocatalytic γ-selective hydrolysis of α,γ-dibenzyl
L
-glutamate, a selective amide hydrolysis of α-benzyl
L
-glutamine, and a selective lactam hydrolysis of alpha-benzyl
L
-pyroglutamate is performed with up to 71% yield.
Semi‐synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7‐aminodesacetoxycephalosporanic acid (7‐ADCA). Recently new routes have been developed which ...allow for the production of adipyl‐7‐ADCA by a novel fermentation process. To complete the biosynthesis of 7‐ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases.
The glutaryl acylase of Pseudomonas SY‐77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase.
In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl‐7‐ADCA by a two‐step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the β‐lactam substrate. The strategy herein described may be generally applicable to all β‐lactam acylases.
Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases ...while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.
Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.
Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.
The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme–substrate complex was determined after soaking crystals of an inactive βN241A ...penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of αF146 and αR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the β-lactam binding site is formed by the side chains of αF146 and βF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of αR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of βQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants αF146Y, αF146A and αF146L all show significant changes in their interaction with the β-lactam substrates as compared with the wild type. The αF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The αF146L and αF146A enzymes had a 3–5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of αF146 in the β-lactam binding site and provide leads for engineering mutants with improved synthetic properties.
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