Elucidation of the stereocontrol mechanisms of the chemical and biosynthetic intramolecular Diels-Alder cycloaddition for the formation of bioactive decalins.

The intramolecular Diels-Alder reaction (IMDA) is a powerful method for regioselective and stereoselective construction of functionalised decalin skeletons, and the recent discovery of enzymes that catalyse IMDA cycloaddition in biosynthesis has generated considerable interest. This study focused on the role of the absolute configuration of the C-6 carbon of the substrate polyene in the stereocontrol of the IMDA reaction catalysed by Fsa2 and Phm7, which construct different enantiomeric decalin skeletons. Their enantiomeric precursor polyenes were synthesised and subjected to enzymatic or thermal IMDA reactions to isolate various diastereomeric decalines and determine their absolute configuration.

Anammox Bacterial -Adenosyl-l-Methionine Dependent Methyltransferase Crystal Structure and Its Interaction with Acyl Carrier Proteins.

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as -adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain.

Diverse reactions catalyzed by cytochrome P450 and biosynthesis of steroid hormone.

Steroid hormones modulate numerous physiological processes in various higher organisms. Research on the physiology, biosynthesis, and metabolic degradation of steroid hormones is crucial for developing drugs, agrochemicals, and anthelmintics. Most steroid hormone biosynthetic pathways, excluding those in insects, have been elucidated, and the roles of several cytochrome P450s (CYPs, P450s), heme (iron protoporphyrin IX)-containing monooxygenases, have been identified.

Structural Basis for the Prenylation Reaction of Carbazole-Containing Natural Products Catalyzed by Squalene Synthase-Like Enzymes.

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole-3,4-quinone-containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well-characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases.

Crystal structure of the adenylation domain from an ε-poly-l-lysine synthetase provides molecular mechanism for substrate specificity.

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain.

Crystal structures of a 6-dimethylallyltryptophan synthase, IptA: Insights into substrate tolerance and enhancement of prenyltransferase activity.

Dimethylallyltryptophan synthases (DMATSs) catalyze the prenyl transfer reaction from dimethylallyl pyrophosphate (DMAPP) to an indole ring. IptA, a member of the DMATS family, is involved in biosynthesis of 6-dimethylallylindole-3-carbaldehyde in Streptomyces sp. SN-593 and catalyzes the C6-prenylation of l-Trp.

Molecular Basis for Two Stereoselective Diels-Alderases that Produce Decalin Skeletons*.

Enzymes catalyzing [4+2] cycloaddition have attracted increasing attention because of their key roles in natural product biosynthesis. Here, we solved the X-ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyze intramolecular [4+2] cycloadditions to form enantiomeric decalin scaffolds during biosynthesis of the HIV-1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin. Computational modeling, using molecular dynamics simulations as well as quantum chemical calculations, demonstrates that the reactions proceed through synergetic conformational constraints assuring transition state-like substrates folds and their stabilization by specific protein-substrate interactions.

Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI.

The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as "micelles." The extracted protein is purified by column chromatography and then is often reconstituted into "liposomes" by removal of the detergent.

Unique features of the ketosynthase domain in a nonribosomal peptide synthetase-polyketide synthase hybrid enzyme, tenuazonic acid synthetase 1.

Many microbial secondary metabolites are produced by multienzyme complexes comprising nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). The ketosynthase (KS) domains of polyketide synthase normally catalyze the decarboxylative Claisen condensation of acyl and malonyl blocks to extend the polyketide chain. However, the terminal KS domain in tenuazonic acid synthetase 1 (TAS1) from the fungus conducts substrate cyclization.

Structural insights into a key step of brassinosteroid biosynthesis and its inhibition.

Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs.

Theoretical Study of Sesterfisherol Biosynthesis: Computational Prediction of Key Amino Acid Residue in Terpene Synthase.

The cyclization mechanisms involved in the biosynthesis of sesterterpenes are not fully understood. For example, there are two plausible reaction pathways for sesterfisherol biosynthesis, which differ in the order of ring cyclization: A-D-B/C (Path a) and A-B-C/D (Path b). It is difficult to capture intermediates of terpene cyclization, which is a complex, domino-type reaction, and so here we employed a combination of experimental and computational methods.

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile sp. A11-2, reveal potential for expanding its substrate selectivity.

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO) during microbial desulfurization.

Crystal structure of D-stereospecific amidohydrolase from Streptomyces sp. 82F2 - insight into the structural factors for substrate specificity.

Unlabelled: D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2, which catalyzes amide bond formation from d-aminoacyl esters and l-amino acids (aminolysis), can be used to synthesize short peptides with a dl-configuration. We found that DAH can use 1,8-diaminooctane and other amino compounds as acyl acceptors in the aminolysis reaction.

The crystal structure of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt): Roles of the axial ligand Tyr75 and two distal arginines in heme binding.

Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule.

Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T.

Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies.

Structural basis for nitrous oxide generation by bacterial nitric oxide reductases.

The crystal structure of the bacterial nitric oxide reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes.

Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus.

The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterized at 2.5 Å resolution.

Characterization and functional modification of StaC and RebC, which are involved in the pyrrole oxidation of indolocarbazole biosynthesis.

The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro.

Molecular structure and function of bacterial nitric oxide reductase.

The crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b(3) and non-heme Fe(B)). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure.

Crystal structure of H2O2-dependent cytochrome P450SPalpha with its bound fatty acid substrate: insight into the regioselective hydroxylation of fatty acids at the alpha position.

Cytochrome P450(SPα) (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H(2)O(2)-dependent P450. P450(SPα) hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450(SPα) with palmitic acid as a substrate at a resolution of 1.

Structural basis of biological N2O generation by bacterial nitric oxide reductase.

Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution.

Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis.

Cytochrome P450(BSβ), a H(2)O(2)-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid-base residue around the heme, which is indispensable for the efficient generation of the active species using H(2)O(2). On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450(BSβ), it was suggested that the role of the general acid-base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450(BSβ) can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid.

Theoretical and experimental studies of the conversion of chromopyrrolic acid to an antitumor derivative by cytochrome P450 StaP: the catalytic role of water molecules.

Chromopyrrolic acid (CPA) oxidation by cytochrome P450 StaP is a key process in the biosynthesis of antitumor drugs (Onaka, H.; Taniguchi, S.; Igarashi, Y.

Electron transfer activation of chromopyrrolic acid by cytochrome p450 en route to the formation of an antitumor indolocarbazole derivative: theory supports experiment.

QM/MM calculations support experiment and show that StaP is a P450 that functions like a peroxidase: its active species is the one-electron-reduced Cpd II species with a radical on CPA, by analogy to cytochrome c peroxidase (CcP), and its reaction with the substrate proceeds by overall proton-coupled electron transfer (PCET), in analogy to the corresponding mechanism in horseradish peroxidase (HRP). The electron transfer is enabled by His250, the presence of carboxylate groups in CPA, and by the H-bonding network that tunes the energetic of the process. Theory supports experiment but reveals some novel aspects of this unusual P450.