Dissection of the Catalytic Mechanisms of Transmembrane Terpene Cyclases Involved in Fungal Meroterpenoid Biosynthesis

Pyr4-family terpene cyclases are noncanonical transmembrane terpene cyclases involved in the biosynthesis of microbial meroterpenoids and catalyze diverse cyclization reactions. Despite the ubiquity of Pyr4-family terpene cyclases in microorganisms, their three-dimensional structures have never been experimentally determined. Herein, we focused on AdrI, the Pyr4-family enzyme for the andrastin A pathway, and its homologues, and performed a series of mutational experiments using their AlphaFold2-generated structures. Intriguingly, we found that AdrI and InsA7, which both accept the same substrate, use different amino acid residues for the initiation of the cyclization cascade. Furthermore, we obtained several AdrI variants with altered product selectivity, one of which dominantly yielded a new meroterpenoid species. Collectively, our study provides important insights into the catalytic functions of Pyr4-family terpene cyclases and will facilitate the engineering of these enzymes.     

Enzymatic synthesis and analytical monitoring of terpene ester by <Superscript>1</Superscript>H NMR spectroscopy

Terpene esters of fatty acids have potential applications in food, cosmetic, and pharmaceutical industries. The present study focuses on the synthesis of terpene esters of long chain fatty acids catalyzed by Candida antarctica lipase B. Different parameters like temperature, solvent, and enzyme concentration for the esterification of terpene alcohols (geraniol and citronellol) with oleic acid were studied. Maximum conversion (98 %) was found for both terpene esters at 60°C in 2,2,4-trimethylpentane as well as in dry hexane and around 95–97 % in other tested solvents. The reaction was also carried out using stearic and linoleic acid in hexane to study the effects of unsaturation in the substrate in which stearic acid showed the maximum conversion. The reaction was monitored by ¹H nuclear magnetic resonance spectroscopy. Using the peak integration values of methylene protons of terpene and terpene ester of δ = 3.6 and 4.0 for citronellol and δ = 4.2 and 4.6 for geraniol, respectively, percentage conversions of each of the esters were calculated.     

The biosynthesis of the fungal meroterpenoids boviquinone-3 and -4 follows two different pathways

A key step in the biosynthesis of the fungal meroterpenoids boviquinone-3 and -4 is the prenylation of 3,4-dihydroxybenzoic acid. In fruit-bodies of Suillus bovinus boviquinone-4 is formed by geranylgeranylation of 3,4-dihydroxybenzoic acid at C-5, whereas in Chroogomphus rutilus the farnesyl side chain of boviquinone-3 is introduced at C-2. The biosynthesis of the terpenoid chain of boviquinone-4 follows the mevalonate route.     

Design and application of basic amino acids displaying enhanced hydrophobicity

Three noncoding basic amino acids, mono-, di-, and trimethyldiaminopropionic acid (mmdap, dmdap, and tmdap), have been synthesized for use in protein design. Covalent modification of a diaminopropionic acid (dap) side chain with an increasing number of methyl moieties results in a family of residues displaying short basic side chains with varying degrees of enhanced hydrophobic character. These residues may be used to introduce charged/polar interactions into the confining hydrophobic interior or interfacial spaces of proteins. As a demonstration of their utility, the ability of these residues to promote interior salt bridge formation at the helix/helix interface of GCN4-p1, a dimeric two-stranded coiled coil, was assessed. Heterodimerization mediated by buried salt bridge formation between a GCN4-based peptide containing either mmdap, dmdap, or tmdap at position 16 and an analogous peptide containing aspartic acid at the same position was studied. Mmdap-derived heterodimers are 0.5 kcal/mol more stable than the corresponding dap-derived heterodimers. This result indicates that the addition of one methyl group to the dap side chain can stabilize the heterodimeric fold. The stabilization can most likely be attributed to a decrease in the desolvation penalty incurred upon folding as well as enhanced van der Waals contacts in the folded state. The addition of three methyl groups to the dap side chain results in heterodimers that are significantly less stable than the corresponding dap-derived heterodimers, suggesting that increased steric bulk is not well accommodated in the interior of this protein. Unexpectedly, the addition of two methyl groups leads to homotrimerization of the dmdap-peptide. The resulting trimer is relatively stable (DeltaG(37)( degrees )(C) degrees = 11.8 kcal/mol) and undergoes cooperative thermal unfolding. The GCN4-p1 system exemplifies how small incremental changes in size and hydrophobicity can alter the folding preferences of a protein. Generally, this versatile suite of residues can be utilized in any protein and offer new options to the protein chemist.     

Simplification of protein NOESY spectra using bioorganic precursor synthesis and NMR spectral editing

A novel method is proposed for the analysis of protein NOEs in solution. In this approach, chemically synthesized precursor compounds for the amino acids valine, leucine, and isoleucine are used for amino acid specific labeling of these hydrophobic residues. The methodology is based on a novel synthetic route to 12C,1H,2H Val, Leu, and Ile side chains selectively labeled with 13CH3 only at the terminal methyl group. In an otherwise 12C,1H labeled protein, discrimination between protons bound to 12C and 13C (or 15N) can be achieved using standard isotope-editing NMR pulse schemes. This strategy significantly relieves problems with spectral overlap through selective observation of interresidue methyl NOEs and will thus be a powerful extension of existing biomolecular NMR methodology.     

Enzymatic Formation of a Skipped Methyl‐Substituted Octaprenyl Side Chain of Longestin (KS‐505a): Involvement of Homo‐IPP as a Common Extender Unit

Longestin (KS‐505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo‐IPP) to afford (3Z)‐3‐methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.     

Capillary electrochromatographic enantioseparations using a packed capillary with a 3 microm OD-type chiral packing

A technique for separating methyl esters of monounsaturated fatty acids by argentation chromatography using silver nitrate-impregnated TLC plates is described. Monounsaturated fatty acid methyl esters are separated from polyunsaturated and saturated fatty acid methyl esters and the monounsaturated fatty methyl esters are resolved according to chain length. cis isomers are well resolved from the corresponding trans isomers. R(F) values for individual monounsaturated fatty acids are very reproducible. The potential of the technique in metabolic studies is demonstrated in the chain elongation of [14C]-18:1(n-9) and delta-9 desaturation of [14C]-18:0 by human skin fibroblasts. Recoveries of individual [14C]-fatty acids for scintillation counting exceed 94%.     

Amphiphilic polymer supports for the asymmetric hydrogenation of amino acid precursors in water

This paper describes the synthesis and characterization of a new class of amphiphilic, water-soluble diblock copolymers based on 2oxazoline derivatives with pendent (2S,4S)-4-diphenylphosphino-2-(diphenylphosphinomethyl)pyrrolidine (PPM) units in the hydrophobic block. The synthetic strategy involves the preparation of a diblock copolymer precursor with ester functionalities in the side chain; which were converted into carboxylic acids in a polymer-analogous step and finally reacted with the PPM ligand. The structures of the copolymers were characterized by (1)H and (31)P NMR spectroscopy and GPC measurements. Subsequently, these polymers were successfully utilized as a polymeric support for the asymmetric hydrogenation of 1) (Z)-alpha-acetamido cinnamic acid and 2) methyl (Z)-alpha-acetamido cinnamate in water, showing 90 % substrate conversion at 25 degrees C within 20 minutes at atmospheric H(2) pressure (1 bar) for methyl (Z)-alpha-acetamido cinnamate.     

Dimericbiscognienynes B and C: New diisoprenyl-cyclohexene-type meroterpenoid dimers from Biscogniauxia sp.

Till now, only three diisoprenyl-cyclohexene/ane-type meroterpenoid dimers have been identified. The isolation of two new diisoprenyl-cyclohexene-type meroterpenoid dimers (dimericbiscognienynes B and C) from Biscogniauxia sp. 71-10-1-1 added new members to this family.     

Isolation, biological significance, synthesis, and cytotoxic evaluation of new natural parathiosteroids A-C and analogues from the soft coral Paragorgia sp

Three unusual new steroid thioesters, parathiosteroids A-C (1a-3a), were isolated from the 2-propanol extract of the soft coral Paragorgia sp. collected in Madagascar. Their structures, determined by detailed spectroscopic analysis, were confirmed by synthesis and represent the first isolation of natural steroids bearing a C22 thioester in their side chain. These compounds displayed cytotoxicity against a panel of three human tumor cell lines at the micromolar level. The preparation of several analogues revealed structure/activity relationships in this type of steroids, for example, that the XCH2CH2NHCOCH3 moiety (X = S, O, NH) in the side chain is essential for the antiproliferative activity, and a low degree of oxidation in the A-ring results in higher bioactivity. These natural products could be biosynthetic intermediates in the steroid side chain degradation pathway involving activation with CoA and beta-oxidations.     

Production of hybrid 16-membered macrolides by expressing combinations of polyketide synthase genes in engineered Streptomyces fradiae hosts

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.     

Autochelation in dipeptide boronic acids: pH-dependent structures and equilibria of Asp-boroPro and His-boroPro by NMR spectroscopy

Many dipeptide boronic acids of the type H(2)N-X-Y-B(OH)(2) are potent protease inhibitors. Interest in these compounds as drugs for cancer, diabetes, and other diseases is growing. Because of the great mutual B-N affinity, cyclization through the N- and B-termini, forming six-membered rings, is a common occurrence at neutral pH and higher where the terminal amino group is unprotonated. Here we report the discovery that when X, the N-terminal amino acid, contains a side chain having a functional group with boron affinity and suitable geometry, additional cyclization in the form of bidentate intramolecular chelation or "autochelation" may occur, predominantly at mid pH. NMR studies of two compounds, l-Aspartyl-l-boroProline (Asp-boroPro) and l-Histidyl-l-boroProline (His-boroPro), are reported here from pH 0.5 to pH 12 by (1)H, (15)N, (13)C, and (11)B NMR. Both of these previously unreported autochelates contain two fused six-membered rings, cis-proline, chiral boron, and -NH(2)(+) protons in slow exchange with water, even at 25 degrees C and pH as high as 4. Using microscopic acid-base equilibrium constants, we show that at high pH (>8 for Asp-boroPro and >10 for His-boroPro) hydroxide competes with the side chains for boron, reducing the chelates from bidentate to monodentate. At low pH (<0.5), proton competition for N-terminal nitrogens causes both compounds to become noncyclic. High chelate stability causes a reduction of the apparent acidic dissociation constant of the protonated N-terminal amino group greater than eight units. In the His-boroPro autochelate, imidazolate anion is produced at the extraordinarily low pH value of approximately 9.     

Synthesis of boronic acid analogues of alpha-amino acids by introducing side chains as electrophiles

A synthetic route has been developed which has allowed us to prepare novel alpha-aminoboronic acids as inhibitors of serine proteases. These compounds were prepared to study the roles of proteases in biological systems. This methodology affords alpha-aminoboronic acids with the general formula R'-NHCH(R)BO(2)-pinanediol, where R = -CH(2)CHF(2), -CH(2)CO(2)tBu, and -(CH(2))(2)CO(2)Me and R' = either H or C(O)R". The latter two compounds are the boronic acid analogues of the natural amino acids aspartic acid and glutamic acid with the side chain carboxylate protected as a tert-butyl or a methyl ester, respectively. Following acylation of the amino group, the side chain tert-butyl ester of boroaspartic acid was removed by treatment with TFA. Boroglutamic acid was obtained as the free boronic acid by hydrolysis with HCl. Prior syntheses of alpha-aminoboronic acids involve the initial addition of an organometallic reagent to a trialkyl borate ester. These conditions do not allow the preparation of compounds with functionalities that are not stable to the strongly basic reaction conditions. The methodology described here allows the preparation of alpha-aminoboronic acids by introducing side chains as electrophiles. This is particularly advantageous for side chains which are prone to elimination or unwanted enolate formation. Specifically, BrCH(2)CHF(2), BrCH(2)COO(t)Bu, and CH(2)=CHCOOMe were allowed to react with the stabilized anion of (phenylthio)methane boronate, PhSCH(2)BO(2)C(6)H(12), to give the substituted boronate. The substituted (phenylthio)methane boronate was converted to the corresponding sulfonium ion by treatment with methyl iodide and subsequently displaced with iodide. The alpha-iodo derivative was converted to the amine by conventional methods.     

Total synthesis of siphonazole and its O-methyl derivative, structurally unusual bis-oxazole natural products

The details of the first syntheses of the unusual bis-oxazole natural products siphonazole and its O-methyl derivative are reported. The cinnamyl substituted oxazole was constructed using diazocarbonyl chemistry, whereby the cinnamamide was reacted with the rhodium carbene derived from methyl 2-diazo-3-oxobutanoate to give a beta-ketoamide that was cyclodehydrated to the corresponding oxazole-4-ester. Reduction to the corresponding aldehyde was followed by coupling with a zinc reagent derived from methyl 2-iodomethyl-5-methyloxazole-4-carboxylate, also prepared using rhodium carbene chemistry, to give, after oxidation of the resulting secondary alcohol, the desired bis-oxazole ketone. The syntheses were completed by hydrolysis of the ester and coupling of the 2,4-pentadienylamine side chain.     

Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3

We demonstrate herein that wild‐type cytochrome P450 BM3 can recognize non‐natural substrates, such as fluorinated C₁₂–C₁₅ chain‐length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non‐fluorinated substrates, spin‐shift measurements suggest that fluoro substituents at the ω‐position can facilitate rearrangement of the dynamic structure of the bulk‐water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis–Menten constant (K_m), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non‐fluorinated counterparts. An enhancement of the turnover frequencies (k_cat) for electron transfer from NADPH to the heme iron and for C? H bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme–substrate (ES state) to the corresponding transition state (ES^≠ state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C₁₂–C₁₅ fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω‐position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12‐fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high‐valent oxoferryl species.     

Theoretical studies of stereoselectivities of intramolecular aldol cyclizations catalyzed by amino acids

The effects of different amino acid catalysts and substrate substituents on the stereoselectivity of the title reactions have been studied with the aid of density functional theory methods. Experimental data available in the literature have been compiled. B3LYP/6-31G(d) calculations match the general experimental trends and provide useful insights into the origins of the variations in stereoselectivities. Acyclic primary amino acids allow a greater conformational flexibility in the aldol transition states compared with proline. This makes them poorer enantioselective catalysts with triketone substrates with a methyl ketone side chain. The steric repulsion upon substitution at the terminal methyl group increases the energy difference between anti- and syn-chairs with primary amino acid catalysts and, consequently, the stereoselectivities. Proline, in contrast, is a poor catalyst for the latter reactions because the substituent's steric bulkiness raises the activation energy of the favored C-C bond-forming pathway.     

Synthesis and Analysis of the Sterically Constrained L-Glutamine Analogues (3S,4R)-3,4-Dimethyl-l-glutamine and (3S,4R)-3,4-Dimethyl-l-Pyroglutamic Acid

The nonproteinogenic amino acids (3S,4R)-3,4-Dimethyl-l-pyroglutamic acid and (3S,4R)-3,4-dimethyl-l-glutamine -- found in the cyclic depsipeptides callipeltin B, callipeltin A, and papuamide A -- were synthesized from a common intermediate derived from l-pyroglutamic acid. The diastereoselective introduction of the methyl groups was accomplished by cuprate addition and enolate alkylation, followed by a kinetic epimerization of the C-4 methyl substituent. (3S,4R)-3,4-dimethyl-l-glutamine shows a conformational restriction of its side chain which may be related to its biological function in the natural products where it is found.     

A highly selective aerobic oxidation process catalyzed by electron-deficient nitroarenes via single electron transfer processes

A versatile and simple method for aerobic oxidation of various aromatics containing an oxygen-functionalized benzylic carbon is reported. Results from oxidation experiments with methyl aryl ketones, benzaldehydes, benzylic alcohols, and mandelic acid are reported; all provide high yields of the corresponding aromatic carboxylic acids. By means of response surface methodology and mechanistic interpretation it is revealed that the oxidation process operates catalytically using electron-deficient nitroarenes, such as 1,3-dinitrobenzene, 1,2,3-trifluoro-4-nitrobenzene, and 2,4-difluoro-1-nitrobenzene, with molecular oxygen as the terminal oxidant. The reaction is carried out in a basic solution of potassium tert-butoxide in tert-butyl alcohol. Since the terminal oxidant is molecular oxygen and only 5-10 mol % of, e.g., 1,3-dinitrobenzene as catalyst is used, the new method represents an environmentally benign alternative to, for example, the well-known haloform reaction. The method is also a convincing alternative when transition metal free conditions are required.     

Optimization of the selectivity of a cyanopropyl stationary phase for the gas chromatographic analysis of trans fatty acids

The quantification of monounsaturated and polyunsaturated trans fatty acids in partially hydrogenated fats by gas-liquid chromatography on a CP-Select CB-FAME capillary column was optimized using equivalent chain length values of fatty acids methyl esters that could coelute in the temperature range from 155 to 200 degrees C. The most appropriate temperature for the simultaneous determination of these trans isomers is around 197 degrees C. It is proposed a system to discriminate trans from cis octadecenoic fatty acid methyl esters based on the angular coefficient values of the equivalent chain length curves. The limits of detection and quantification were 0.28 and 0.93 mg g(-1). Quantification was performed in the range from 0.93 to 280 mg g(-1). Quantification accuracy was estimated by spike recovery of elaidic and trans-13-octadecenoic acids in hydrogenated fat samples. The obtained levels were from 97.60 to 103.28% for elaidic acid and from 98.12 to 99.27% for trans-13-octadecenoic acid. These results indicate that the accuracy of the methodology proposed for the quantification of monounsaturated and polyunsaturated trans fatty acids in hydrogenated fats is adequate.