Jennifer N. Andexer

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

How to overcome limitations in biotechnological processes - examples from hydroxynitrile lyase applications

During the last decades, enzymes became very versatile catalysts for a variety of reactions including natural and unnatural compounds. However, many enzyme-catalysed reactions suffer from diverse restrictions because of limitations related to process parameters or the enzyme. The understanding and overcoming of those undesired side effects is therefore mandatory for the implementation of optimal process parameters. To achieve this aim, various methods from molecular biology and reaction engineering can be employed. By focusing on the hydroxynitrile lyase-catalysed synthesis of enantiopure cyanohydrins, we give an overview of strategies to improve commercially utilized enzymes and to suppress non-enzymatic reactions. Particular emphasis is placed on the necessity to combine approaches from different fields, such as enzyme engineering and reaction engineering.

Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with α/β-hydrolase fold.

Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL.

Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

Complex reduced polyketides comprise an abundant class of microbial natural products with an impressive range of chemical and functional diversity. They are synthesised on giant modular polyketide synthases (PKSs) in an assembly-line process in which each successive module of enzymatic domains normally catalyses a single cycle of chain extension and modification before the growing polyketide chain is passed on. The chemical nature of the extender unit, its stereochemistry and its level of reduction are specified by the type of domains present in each module. The initially formed b-ketoacyl thioester can either be retained or reduced to a b-hydroxyacyl-, 2,3enoylor saturated acyl thioester intermediate, depending on the presence of ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains, respectively. Significant progress has been made in defining the respective contributions of each type of reductive domain (KR, DH, ER) to this outcome. However, the mechanisms by which certain structural features arise remain poorly understood. For example, most double bonds in these compounds (whether retained or further reduced) have trans (E) geometry, arising from the syn dehydration of 3R alcohols (as in the analogous process in fatty acid synthesis) 6] but a significant fraction of polyketides have double bonds with the alternative cis (Z) configuration. In some cases, cis double bonds have been shown to arise from the action of exogenous enzymes, but for other modular PKSs it has been proposed that cis double bonds arise by syn dehydration of a 3S alcohol by the DH domain. This hypothesis implies that in such cases the double bond geometry is controlled by the stereochemical outcome of KR-catalysed ketoreduction, rather than directly by the DH itself, and so cisgenerating modules should contain KR domains predicted to dictate production of 3S alcohols. 11] This correlation often holds true, but known exceptions include cis double bonds in the antibiotics rifamycin, halstoctacosanolide and thuggacin, in the anticancer compound phoslactomycin, and in the anti-angiogenic compound borrelidin (see the Supporting Information). We report here the recombinant expression of individual enzymatically active DH domains from modules 2 and 3 of the borrelidin PKS. We show that, at least when acting on surrogate substrates in vitro, the apparently cis-producing DH domain from module 3 follows exactly the same stereochemical course as the trans-producing DH domain of the preceding module 2, accepting 3R but not 3S alcohol substrates and carrying out syn elimination. The formation of the cis double bond in borrelidin is therefore very unlikely to be determined solely by the stereochemical course of the preceding ketoreduction. Borrelidin (1), an unusual polyketide with potent cytotoxic, anti-angiogenic and anti-malarial activities, contains both a trans and a cis double bond, as confirmed by total synthesis and X-ray diffraction analysis. Both bor modules 2 (trans double bond) and 3 (cis double bond) contain the expected set of reducing domains (KR and DH) (Figure 1). Both bor KR domains are strongly predicted, on the basis of the presence of characteristic active site sequence motifs, to produce 3R alcohol products, and when a methyl branch is present at the a-position a 2R,3R configuration. On this basis, the current model for dehydration would suggest that each of BorDH2 and BorDH3 should yield a trans double bond. X-ray crystal structures are available for DH domains from the erythromycin PKS and from the curacin PKS, and these show very close similarity in overall fold and in active site architecture to each other and to the DH domain of animal fatty acid synthase. The solved structures contain no bound ligand, but modelling has indicated that the active site could in principle accommodate syn elimination to give either trans or cis geometry. We wished to determine whether, despite the sequencebased KR prediction, the actual substrate for BorDH3 might be the 3S isomer. We have expressed and purified both BorDH2 and BorDH3 as individual proteins in Escherichia coli (Supporting Information), and have assayed them for activity against the N-acetylcysteamine (SNAC) thioesters of (3R)and (3S)-3hydroxybutanoic acid (2, Figure 2), as surrogate substrates for the normal acylcarrier protein (ACP) bound intermediates on the PKS. As expected, BorDH2 converted (3R)-2 exclusively into crotonyl-SNAC (trans-3). The trans configuration was confirmed by NMR analysis (Supporting Information). In contrast, (3S)-2 was not a substrate. The degrees of conversion observed were low (20–30 % after 16 h), but fully reproducible. Successful conversion required substantial amounts of enzyme (up to 8 mg mL ) relative to previous studies using ACP-bound sub[a] Dr. O. Vergnolle, Dr. F. Hahn, Dr. A. Baerga-Ortiz, Prof. Dr. P. F. Leadlay, Dr. J. N. Andexer Department of Biochemistry, University of Cambridge 80 Tennis Court Road, Cambridge, CB21GA (UK) Fax: (+ 44) 1223-766002 E-mail : jennifer.andexer@pharmazie.uni-freiburg.de [b] Dr. O. Vergnolle Present address: Biology Department, Brooklyn College City University of New York 2900 Bedford Avenue, Brooklyn, NY 11210 (USA) [c] Dr. A. Baerga-Ortiz Present address: School of Medicine, University of Puerto Rico San Juan (Puerto Rico) [d] Dr. J. N. Andexer Present address: Pharmaceutical and Medicinal Chemistry University of Freiburg Albertstrasse 25, 79104 Freiburg (Germany) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100011.

Hydroxynitrile Lyases with α/β‐Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R‐selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β‐hydrolase fold, very similar to the homologous, but S‐selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser‐His‐Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)‐ and (S)‐mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)‐mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main‐chain amide groups and an α‐helix dipole very similar to α/β‐hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.

A high-throughput screening assay for hydroxynitrile lyase activity

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.

Uncovering the origin of Z-configured double bonds in polyketides: intermediate E-double bond formation during borrelidin biosynthesis

Formation of Z-configured double bonds in reduced polyketides is uncommon and their origins have not been extensively studied. To investigate the origin of the Z-configured double bond in the macrolide borrelidin, the recombinant dehydratase domains BorDH2 and BorDH3 were assayed with a synthetic analogue of the predicted tetraketide substrate. The configuration of the dehydrated products was determined to be E in both cases by comparison to synthetic standards. Detailed NMR spectroscopic analysis of the biosynthetic intermediate pre-borrelidin confirmed the E,E-configuration of the full-length polyketide synthase product. In contrast to a previously-proposed hypothesis, our results show that in this case the Z-configured double bond is not formed via dehydration from a 3 L-configured precursor, but rather as the result of a later isomerization process.

Asymmetric C‐Alkylation by the S‐Adenosylmethionine‐Dependent Methyltransferase SgvM

S-Adenosylmethionine-dependent methyltransferases (MTs) play a decisive role in the biosynthesis of natural products and in epigenetic processes. MTs catalyze the methylation of heteroatoms and even of carbon atoms, which, in many cases, is a challenging reaction in conventional synthesis. However, C-MTs are often highly substrate-specific. Herein, we show that SgvM from Streptomyces griseoviridis features an extended substrate scope with respect to the nucleophile as well as the electrophile. Aside from its physiological substrate 4-methyl-2-oxovalerate, SgvM catalyzes the (di)methylation of pyruvate, 2-oxobutyrate, 2-oxovalerate, and phenylpyruvate at the β-carbon atom. Chiral-phase HPLC analysis revealed that the methylation of 2-oxovalerate occurs with R selectivity while the ethylation of 2-oxobutyrate with S-adenosylethionine results in the S enantiomer of 3-methyl-2-oxovalerate. Thus SgvM could be a valuable tool for asymmetric biocatalytic C-alkylation reactions.

Mechanistic Implications for the Chorismatase FkbO Based on the Crystal Structure

Chorismate-converting enzymes are involved in many biosynthetic pathways leading to natural products and can often be used as tools for the synthesis of chemical building blocks. Chorismatases such as FkbO from Streptomyces species catalyse the hydrolysis of chorismate yielding (dihydro)benzoic acid derivatives. In contrast to many other chorismate-converting enzymes, the structure and catalytic mechanism of a chorismatase had not been previously elucidated. Here we present the crystal structure of the chorismatase FkbO in complex with a competitive inhibitor at 1.08Å resolution. FkbO is a monomer in solution and exhibits pseudo-3-fold symmetry; the structure of the individual domains indicates a possible connection to the trimeric RidA/YjgF family and related enzymes. The co-crystallised inhibitor led to the identification of FkbOs active site in the cleft between the central and the C-terminal domains. A mechanism for FkbO is proposed based on both interactions between the inhibitor and the surrounding amino acids and an FkbO structure with chorismate modelled in the active site. We suggest that the methylene group of the chorismate enol ether takes up a proton from an active-site glutamic acid residue, thereby initiating chorismate hydrolysis. A similar chemistry has been described for isochorismatases, albeit implemented in an entirely different protein scaffold. This reaction model is supported by kinetic data from active-site variants of FkbO derived by site-directed mutagenesis.

Regiocomplementary O-Methylation of Catechols by Using Three-Enzyme Cascades

S‐Adenosylmethionine (SAM)‐dependent enzymes have great potential for selective alkylation processes. In this study we investigated the regiocomplementary O‐methylation of catechols. Enzymatic methylation is often hampered by the need for a stoichiometric supply of SAM and the inhibitory effect of the SAM‐derived byproduct on most methyltransferases. To counteract these issues we set up an enzyme cascade. Firstly, SAM was generated from l‐methionine and ATP by use of an archaeal methionine adenosyltransferase. Secondly, 4‐O‐methylation of the substrates dopamine and dihydrocaffeic acid was achieved by use of SafC from the saframycin biosynthesis pathway in 40–70 % yield and high selectivity. The regiocomplementary 3‐O‐methylation was catalysed by catechol O‐methyltransferase from rat. Thirdly, the beneficial influence of a nucleosidase on the overall conversion was demonstrated. The results of this study are important milestones on the pathway to catalytic SAM‐dependent alkylation processes.