Enzymatic Baeyer–Villiger oxidation of steroids with cyclopentadecanone monooxygenase

Recombinant cyclopentadecanone monooxygenase from Pseudomonas sp. catalyzed the preparative-scale Baeyer–Villiger oxidation of numerous 3 and 17-ketosteroids with a full control of the regiochemistry of the produced lactones. The recovered product yields were up to 42%.     

Expression and metabolic activity of flavin‐containing monooxygenase 1 in cynomolgus macaque kidney

Flavin‐containing monooxygenase 1 (FMO1) largely remains to be characterized in cynomolgus macaque kidney. Immunoblotting showed expression of cynomolgus FMO1 in kidneys where activities of FMO1 (benzydamine N‐oxygenation) were detected. No sex differences were observed in their contents or activities. These results suggest the functional role of cynomolgus FMO1 in kidney.     

Microbial Baeyer–Villiger oxidation of 4,4-disubstituted cyclohexan- and cyclohexenones by recombinant whole-cells expressing monooxygenases of bacterial origin

Screening of 4,4-disubstituted and 3,4,5-polysubstituted cyclohexan- and cyclohexenones with eight different overexpression systems of microbial monooxygenases in recombinant Escherichia coli provided valuable information about substrate acceptance and enantioselectivity of this enzyme family, which are responsible for the stereoselective Baeyer–Villiger biooxidation of ketones. For this purpose whole-cell mediated biotransformations were realized to overcome some limitations in the application of cofactor dependent biocatalysts. The different behavior of various enzymes reflects a recent hypothesis about two distinct clusters of biooxidation catalysts. In contrast to isolated enzyme biooxidations, recombinant cells did not yield unsaturated lactone products derived from cycloalkenones. They rather displayed reductase activity to reduce such precursors to saturated ketones, which were subsequently oxidized to the corresponding Baeyer–Villiger products in a sequential two-step biotransformation.     

Cofactor‐induced reversible folding of Flavodoxin‐4 from Lactobacillus acidophilus

Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN‐binding mechanism to flavodoxins‐4 were obtained from the NMR structures of the apo‐protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO ) and exhibits the characteristic flavodoxin fold, with a central five‐stranded parallel β–sheet and five α‐helices forming an α/β‐sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the β‐sheet and covers the FMN‐binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.     

Crystal structure of AdoMet radical enzyme 7‐carboxy‐7‐deazaguanine synthase from Escherichia coli suggests how modifications near [4Fe–4S] cluster engender flavodoxin specificity

7‐Carboxy‐7‐deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6‐carboxy‐5,6,7,8‐tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7‐deazaguanine natural products. QueE is a member of the S‐adenosyl‐L‐methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX₃CX?C motif, to bind a [4Fe‐4S] cluster, and a partial (β/α)₆ TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster‐binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re‐haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin. Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7‐carboxy‐7‐deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.     

Synthesis and cytotoxic activity of some novel polycyclic gamma-butyrolactones

Baeyer–Villiger oxidation of 5-aryl-7,11,11-trimethyltricyclo[5.4.0.0^3,6]-undec-1-en-4-ones 4a–h by H₂O₂ and formic acid in methanol yields mixtures of 3b,7,7-trimethyl-3-phenyl-3,3a,3b,4,5,6,7,8a-octahydro-1H-indeno-[1,2-c]furan-1-ones 8a–h and 3b,7,7-trimethyl-3-phenyl-3,3a,3b,4,5,6,7,8a-octahydro-1H-indeno-[1,2-c]furan-2-ones 9a–h in high yields. The obtained butyrolactones 8a–h display cytotoxic activity against a number of human cancer cells.     

Crystal structure of a putative pyridoxine 5′‐phosphate oxidase (Rv2607) from Mycobacterium tuberculosis

The three‐dimensional structure of Rv2607, a putative pyridoxine 5′‐phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X‐ray crystallography to 2.5 Å resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

Analysis of the sodium chloride‐dependent respiratory kinetics of wheat mitochondria reveals differential effects on phosphorylating and non‐phosphorylating electron transport pathways

A number of previous studies have documented the gross response of mitochondrial respiration to salinity treatment, but it is unclear how NaCl directly affects the kinetics of plant phosphorylating and non‐phosphorylating electron transport pathways. This study investigates the direct effects of NaCl upon different respiratory pathways in wheat, by measuring rates of isolated mitochondrial oxygen consumption across different substrate oxidation pathways in saline media. We also profile the abundance of respiratory proteins by using targeted selected reaction monitoring (SRM) mass spectrometry of mitochondria isolated from control and salt‐treated wheat plants. We show that all pathways of electron transport were inhibited by NaCl concentrations above 400 mM; however electron transfer chains showed divergent responses to NaCl concentrations between 0 and 200 mM. Stimulation of oxygen consumption was measured in response to NaCl in scenarios where exogenous NADH was provided as substrate and electron flow was coupled to the generation of a proton gradient across the inner membrane. Protein abundance measurements show that several enzymes with activities less affected by NaCl are induced by salinity, whereas enzymes with activities inhibited by NaCl are depleted. These data deepen our understanding of how plant respiration responds to NaCl, offering new mechanistic explanations for the divergent salinity responses of whole‐plant respiratory rate in the literature.     

Effects of lysophospholipids on the generation of reactive oxygen species by fMLP‐ and PMA‐stimulated human neutrophils

In this study, the effects of exogenous lysophospholipids—lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine—on the kinetics of reactive oxygen species (ROS) production by human neutrophils are described. The ROS production by human neutrophils was monitored by luminol‐amplified chemiluminescence after cell stimulation with the chemotactic tripeptide, fMLP, or with the phorbol ester, PMA. The interaction of lysophospholipids with the membrane of human neutrophils was additionally tested by mass spectrometry. Lysophosphatidylcholine showed the most pronounced effect on the chemiluminescence pattern, as well as the intensity of the fMLP and PMA‐stimulated cells, whereas lysophosphatidic acid showed a slight priming effect when fMLP was used for stimulation. In the case of fMLP‐stimulated cells, lysophosphatidylcholine inhibited the first phase and enhanced the second phase of chemiluminescence, whereas the chemiluminescence of PMA‐stimulated neutrophils was inhibited in a concentration‐dependent manner. We conclude that lysophosphatidylcholine is able to interact with protein kinase C‐dependent signalling pathways leading to NADPH oxidase activation. Copyright © 2002 John Wiley & Sons, Ltd.     

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ‐lyase based on substrate‐assisted catalysis

Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.     

Genetic diversity and recombination in natural populations of the nematode‐trapping fungus Arthrobotrys oligospora from China

Nematophagous fungi can trap and capture nematodes and other small invertebrates. This unique ability has made them ideal organisms from which to develop biological control agents against plant‐ and animal‐parasitic nematodes. However, effective application of biocontrol agents in the field requires a comprehensive understanding about the ecology and population genetics of the nematophagous fungi in natural environments. Here, we genotyped 228 strains of the nematode‐trapping fungus Arthrobotrys oligospora using 12 single nucleotide polymorphic markers located on eight random DNA fragments. The strains were from different ecological niches and geographical regions from China. Our analyses identified that ecological niche separations contributed significantly, whereas geographic separation contributed relatively little to the overall genetic variation in our samples of A. oligospora. Interestingly, populations from stressful environments seemed to be more variable and showed more evidence for recombination than those from benign environments at the same geographic areas. We discussed the implications of our results to the conservation and biocontrol application of A. oligospora in agriculture and forestry.