Protein engineering of oxidosqualene-lanosterol cyclase into triterpene monocyclase.

A computational modeling/protein engineering approach was applied to probe H234, C457, T509, Y510, and W587 within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7), which spatially affects the C-10 cation of lanosterol formation. Substitution of Trp587 to aromatic residues supported the "aromatic hypothesis" that the π-electron-rich pocket is important for the stabilization of electron-deficient cationic intermediates. The Cys457 to Gly and Thr509 to Gly mutations disrupted the pre-existing H-bond to the protonating Asp456 and the intrinsic His234 : Tyr510 H-bond network, respectively, and generated achilleol A as the major product. An H234W/Y510W double mutation altered the ERG7 function to achilleol A synthase activity and generated achilleol A as the sole product. These results support the concept that a few-ring triterpene synthase can be derived from polycyclic cyclases by reverse evolution, and exemplify the power of computational modeling coupled with protein engineering both to study the enzyme's structure-function-mechanism relationships and to evolve new enzymatic activity.

Protein engineering of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase into parkeol synthase.

A Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase mutant, ERG7(T384Y/Q450H/V454I), produced parkeol but not lanosterol as the sole end product. Parkeol undergoes downstream metabolism to generate compounds 9 and 10. In vitro incubation of parkeol produced a product profile similar to that of the in vivo experiment. In summary, parkeol undergoes a metabolic pathway similar to that of cycloartenol in yeast but distinct from that of lanosterol in yeast, suggesting that two different metabolic pathways of postoxidosqualene cyclization may exist in S. cerevisiae.

The cysteine 703 to isoleucine or histidine mutation of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae generates an iridal-type triterpenoid.

The Cys703 to Ile or His mutation within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase ERG7 (ERG7(C703I/H)) generates an unusual truncated bicyclic rearranged intermediate, (8R,9R,10R)-polypoda-5,13E,17E,21-tetraen-3ß-ol, related to iridal-skeleton triterpenoid. Numerous oxidosqualene-cyclized truncated intermediates, including tricyclic, unrearranged tetracyclic with 17α/ß exocyclic hydrocarbon side chain, rearranged tetracyclic, and chair-chair-chair tricyclic intermediates (compounds 3-9), were also isolated from the ERG7(C703X) site-saturated mutations or the ERG7(F699T/C703I) double mutation, indicating the functional role of the Cys703 residue in stabilizing the bicyclic C-8 cation and the rearranged intermediate or interacting with Phe699, and opened a new avenue of engineering ERG7 for producing biological active agents.

Photoactivation studies of zinc porphyrin-myoglobin system and its application for light-chemical energy conversion.

An artificial zinc porphyrin-myoglobin-based photo-chemical energy conversion system, consisting of ZnPP-Mb or ZnPE(1)-Mb as a photosensitizer, NADP(+) as an electron acceptor, and triethanolamine as an electron donor, has been constructed to mimic photosystem I. The photoirradiated product is able to reduce a single-electron acceptor protein cytochrome c, but cannot catalyze the two-electron reduction of acetaldehyde by alcohol dehydrogenase, thus demonstrating a single electron transfer mechanism. Furthermore, the artificial system can bifunctionally promote oxidoredox reactions, depending on the presence or absence of a sacrificial electron donor, thus suggesting its potential application in electrochemical regeneration steps involved in chemical transformation and/or energy conversion.

Purification and properties of a novel phenolic antioxidant from Radix astragali fermented by Aspergillus oryzae M29.

The Chinese herb Radix astragalus (RA) has been widely used as a dietary supplement in Asia, and there are numerous reports on its bioactivities. However, there are no reports to date regarding the use of Aspergillus spp. in the culture medium of the RA plant for the production of phenolic antioxidants. In this study, utilizing the fungus Aspergillus to ferment the native RA has successfully resulted in a significant increase in the phenolic contents of RA, and the fermented RA also revealed much better antioxidant activity toward 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, hydroxyl radical, superoxide radical and peroxyl radical than those of unfermented RA. Among these phenolics, a potent novel antioxidant was isolated and identified as 3,4-di(4'-hydroxyphenyl) isobutyric acid with a molecular weight of 272, by ESI-MS (electrospray ionization mass), ¹H NMR (nuclear magnetic resonance), ¹³C NMR, DEPT (distortionless enhancement by polarization transfer)-NMR, HMQC (heteronuclear multiple quantum coherence), and HMBC (heteronuclear multiple bond correlation) spectra. These data demonstrated that the solid-state bioprocessing strategy could be an innovative approach to enhance the antioxidant activity of RA.

Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense.

The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-L-glutaminylglycine as a substrate, revealed apparent K(m) and k(cat)/K(m) values of 52.66 mM and 40.42 mM(-1) min(-1), respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC(50) values of 0.75 and 0.65 mM, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of (N)SQAETQ(C)-C-DNS and C-DNS-(N)QR(C) sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition.

Mutation of isoleucine 705 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae affects lanosterol's C/D-ring cyclization and 17α/ß-exocyclic side chain stereochemistry.

Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/ß-exocyclic side chain stereochemistry.

Crystal structure and mutational analysis of aminoacylhistidine dipeptidase from Vibrio alginolyticus reveal a new architecture of M20 metallopeptidases.

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.

Alteration of the substrate's prefolded conformation and cyclization stereochemistry of oxidosqualene-lanosterol cyclase of Saccharomyces cerevisiae by substitution at phenylalanine 699.

The Saccharomyces cerevisiae ERG7(Phe699) mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated tricyclic compounds, one tetracyclic 17alpha-exocyclic unrearranged intermediate, and two 17beta-exocyclic truncated rearranged intermediates. These results provided direct evidence for the importance of the residue in affecting mechanistic transitions between C-B-C and C-C-C substrate conformation and between the 17alpha- and 17beta-exocyclic side chain stereochemistry as well as in stabilizing the 6-6-5 tricyclic and the protosteryl C-17 cations.

Importance of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase tyrosine 707 residue for chair-boat bicyclic ring formation and deprotonation reactions.

A contact mapping strategy was applied to identify putative amino acid residues that influence the oxidosqualene-lanosterol B-ring cyclization reaction. A bicyclic intermediate with two altered deprotonation products, in conjunction with lanosterol, were isolated from the ERG7(Y707X) mutants, indicating that the Tyr707 residue may play a functional role in stabilizing the chair-boat bicyclic C-8 cation and the lanosteryl C-8/C-9 cation intermediates.

Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase: a chemistry-biology interdisciplinary study of the protein's structure-function-reaction mechanism relationships.

The oxidosqualene cyclases (EC 5.4.99-) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)-2,3-oxidosqualene into a distinct array of sterols and triterpenes. The relationship between the cyclization mechanism and the enzymatic structure is extremely complex and compelling. This review covers the historical achievements of biomimetic studies and current progress in structural biology, molecular genetics, and bioinformatics studies to elucidate the mechanistic and structure-function relationships of the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase-catalyzed cyclization/rearrangement reaction.

Protein plasticity: a single amino acid substitution in the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase generates protosta-13(17),24-dien-3beta-ol, a rearrangement product.

To provide insights into the structure-function relationships of oxidosqualene-lanosterol cyclase (ERG7) from Saccharomyces cerevisiae, the Phe699 was exchanged against hydrophilic polar uncharged residues Ser, Thr, Cys, Gln, and Tyr to characterize its product profile and functional role in ERG7 activity. Among the substitutions, only the ERG7(F699T) mutant produced novel protosta-13(17),24-dien-3beta-ol as the sole truncated rearrangement product. The results suggest that Phe699Thr mutation is likely to affect the C-17 cation stabilization during the rearrangement process.

Phenylalanine 445 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences C-Ring cyclization and deprotonation reactions.

[reaction: see text] We describe the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation-pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.

Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions.

Site-saturated mutagenesis experiments were carried out on the His234 residue of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) to characterize its functional role in ERG7 activity and to determine its effect on the oxidosqualene cyclization/rearrangement reaction. Two novel intermediates, (13alphaH)-isomalabarica-14(26),17E,21-trien-3beta-ol and protosta-20,24-dien-3beta-ol, isolated from ERG7(H234X) mutants, provided direct mechanistic evidence for formation of the chair-boat 6-6-5 tricyclic Markovnikov cation and protosteryl cation that were assigned provisionally to the ERG7-catalyzed biosynthetic pathway. In addition, we obtained mutants that showed a complete change in product specificity from lanosterol formation to either protosta-12,24-dien-3beta-ol or parkeol production. Finally, the repeated observation of multiple abortive and/or alternative cyclization/arrangement products from various ERG7(H234X) mutants demonstrated the catalytic plasticity of the enzyme. Specifically, subtle changes in the active site affect both the stability of the cation-pi interaction and generate product diversity.

Purification, tandem mass characterization, and inhibition studies of oxidosqualene-lanosterol cyclase enzyme from bovine liver.

The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been isolated from the microsomal membrane fraction and purified to homogeneity by ultracentrifugation, Q-Sepharose, hydroxyapatite, and HiTrap heparin chromatographies. The purified protein required Triton X-100 to retain its highest activity. The cyclase had a molecular mass of approximately 70 and approximately 140 kDa, as evidenced by a single protein band on silver-stained SDS-PAGE and Coomassie-stained PAGE, respectively. Results from Edman degradation of OSC suggested that it might have a blocked N-terminus. Further peptide mapping coupled with tandem mass spectrometric determination identified three peptide fragments, ILGVGPDDPDLVR, LSAEEGPLVQSLR, and NPDGGFATYETK, which are highly homologous to human, rat, and mouse OSCs. The purified cyclase showed pH and temperature optima at pH 7.4 and 37 degrees C, respectively. The apparent K(M) and k(cat)/K(M) values were estimated to be 11 microM and 1.45 mM(-1)min(-1), respectively. Inhibition studies using both Ro48-8071 and N-(4-methylenebenzophenonyl)pyridinium bromide showed potent inhibition of OSC with an IC(50) of 11 nM and 0.79 microM, respectively. Results from DTNB modification and DTNB coupled with Ro48-8071 competition study suggest that two sulfhydryl groups are involved in the catalysis but not located in the substrate binding pocket or catalytic active site. The purified OSC was maximally inactivated by diethyl pyrocarbonate near neutral pH and re-activated by hydroxylamine, indicating the modification of histidine residues. The stoichiometry of histidine modification and the extent of inactivation showed that two essential histidine residues per active site are necessary for complete bovine liver OSC activity.