SNPs in cytochrome P450 genes decide on the fate of individuals with genetic predisposition to Parkinson's disease.

Parkinson's disease (PD) is one of the most frequent neurological diseases affecting millions of people worldwide. While the majority of PD cases are of unknown origin (idiopathic), about 5%-10% are familial and linked to mutations in different known genes. However, there are also people with a genetic predisposition to PD who do not develop the disease. To elucidate factors leading to the manifestation of PD we compared the occurrence of single nucleotide polymorphisms (SNPs) in various cytochrome P450 (P450) genes in people with a genetic predisposition and suffering from PD (GPD) to that of people, who are genetically predisposed, but show no symptoms of the disease (GUN). We used the PPMI (Parkinson's Progression Markers Initiative) database and the gene sequences of all 57 P450s as well as their three redox partners. Corresponding odds ratios (OR) and confidence intervals (CI) were calculated to assess the incidence of the various SNPs in the two groups of individuals and consequently their relation to PD. We identified for the first time SNPs that are significantly (up to 10fold!) over- or under-represented in GPD patients compared to GUN. SNPs with OR > 5 were found in 10 P450s being involved in eicosanoid, vitamin A and D metabolism as well as cholesterol degradation pointing to an important role of endogenous factors for the manifestation of PD clinical symptoms. Moreover, 12 P450s belonging to all P450 substrate classes as well as POR have SNPs that are significantly under-represented (OR < 0.2) in GPD compared to GUN, indicating a protective role of those SNPs and the corresponding P450s regarding disease advancement. To the best of our knowledge our data for the first time demonstrate an association between known PD predisposition genes and SNPs in other genes, shown here for different P450 genes and for their redox partner POR, which promote the manifestation of the disease in familial PD. Our results thus shed light onto the pathogenesis of PD, especially the switch from GUN to GPD and might further help to advance novel strategies for preventing the development or progression of the disease.

Protocol to identify amino acids bound to tRNA by aminoacylation using mass spectrometry.

tRNA-bound amino acids often need to be identified, for instance, in cases where different amino acids compete for binding to the same tRNA. Here, we present a mass-spectrometry-based protocol to determine the amino acids bound to tRNA by aminoacylation. We detail how to perform the aminoacylation reaction, the preparation of the aminoacyl-tRNA for measurement, and the mass spectrometric analysis. We use arginine acylation as an example; however, this protocol can be applied to any other amino acid.

Regio- and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X-ray crystallography and computational modeling.

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio- and stereoselective manner to 16ß-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme-iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon-hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis toward improved enzymatic properties of this enzyme.

A CYPome-wide study reveals new potential players in the pathogenesis of Parkinson's disease.

Genetic and environmental factors lead to the manifestation of Parkinson's disease (PD) but related mechanisms are only rudimentarily understood. Cytochromes P450 (P450s) are involved in the biotransformation of toxic compounds and in many physiological processes and thus predestinated to be involved in PD. However, so far only SNPs (single nucleotide polymorphisms) in CYP2D6 and CYP2E1 have been associated with the susceptibility of PD. Our aim was to evaluate the role of all 57 human P450s and their redox partners for the etiology and pathophysiology of PD and to identify novel potential players which may lead to the identification of new biomarkers and to a causative treatment of PD. The PPMI (Parkinson's Progression Markers Initiative) database was used to extract the gene sequences of all 57 P450s and their three redox partners to analyze the association of SNPs with the occurrence of PD. Applying statistical analyses of the data, corresponding odds ratios (OR) and confidence intervals (CI) were calculated. We identified SNPs significantly over-represented in patients with a genetic predisposition for PD (GPD patients) or in idiopathic PD (IPD patients) compared to HC (healthy controls). Xenobiotic-metabolizing P450s show a significant accumulation of SNPs in PD patients compared with HC supporting the role of toxic compounds in the pathogenesis of PD. Moreover, SNPs with high OR values (>5) in P450s catalyzing the degradation of cholesterol (CYP46A1, CY7B1, CYP39A1) indicate a prominent role of cholesterol metabolism in the brain for PD risk. Finally, P450s participating in the metabolism of eicosanoids show a strong over-representation of SNPs in PD patients underlining the effect of inflammation on the pathogenesis of PD. Also, the redox partners of P450 show SNPs with OR > 5 in PD patients. Taken together, we demonstrate that SNPs in 26 out of 57 P450s are at least 5-fold over-represented in PD patients suggesting these P450s as new potential players in the pathogenesis of PD. For the first time exceptionally high OR values (up to 12.9) were found. This will lead to deeper insight into the origin and development of PD and may be applied to develop novel strategies for a causative treatment of this disease.

Resurrection and characterization of ancestral CYP11A1 enzymes.

Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.

Metabolism of oral turinabol by the human brain cholesterol 24-hydroxylase CYP46A1.

The human microsomal cytochrome P450 enzyme CYP46A1 plays a crucial role in cholesterol elimination from the brain. It performs a 24-hydroxylation of cholesterol and is of outstanding significance for memory and cognition. This study demonstrates the catalytic activity of human CYP46A1 towards an anabolic androgenic steroid, oral turinabol (dehydrochloromethyltestosterone, 4-chloro-17ß-dihydroxy,17α-methylandrosta-1,4-dien-3-one), which is a doping substance. CYP46A1 is the first human microsomal steroid-converting P450 showing activity towards this xenobiotic compound. Furthermore, the inhibitory effect of oral turinabol on the cholesterol conversion has been investigated in vitro demonstrating competition of the two substrates on the active site of CYP46A1 which might be of importance for potential pathogenic effects of oral turinabol. The conversion of oral turinabol was found to be selective resulting in the formation of only one product, as shown by HPLC analysis. To produce sufficient amounts of this product for NMR analysis, a system expressing human full-length CYP46A1 and CPR on a bicistronic vector was successfully developed realizing the selective cholesterol 24-hydroxylation in E. coli in mg amounts. Using this novel whole-cell system, the conversion of oral turinabol was performed and the product of this conversion by CYP46A1 was isolated and identified as 16ß-hydroxy oral turinabol by NMR.

Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of l-lysine by l-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational l-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinked.

Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium.

Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous ß-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems.

Cell-free transcription-translation systems are a versatile tool to study gene expression, enzymatic reactions and biochemical regulation mechanisms. Because cell-free transcription-translation systems are often derived from cell lysates, many different substances, among them amino acids, are present. However, experiments concerning the incorporation of non-canonical amino acids into proteins require a system with negligible amounts of canonical analogs. Here we propose a two-step method for the removal of residual free lysine in an all Escherichia coli-based cell-free expression system. The first step consists of the expression of a high-lysine dummy protein. The second step consists of direct removal via binding between lysine and DNA. The presented method is an efficient, fast and simple way to remove residual lysine without altering the system ability to perform gene expression.

High-yield C11-oxidation of hydrocortisone by establishment of an efficient whole-cell system in Bacillus megaterium.

Steroidal compounds are one of the most widely marketed pharmaceutical products. Chemical synthesis of steroidal compounds faces many challenges, including the requirement for multiple chemical steps, low yield and selectivity in several synthesis steps, low profitability and the production of environmental pollutants. Consequently, in recent decades there has been growing interest in the use of microbial systems to produce pharmaceutical compounds. Several microbial systems have recently been developed for the microbial synthesis of the glucocorticoid hydrocortisone, which serves as a key intermediate in the production of several other pharmaceutically important steroidal compounds. In this study, we sought to establish an efficient, microbial-based system, for the conversion of hydrocortisone into cortisone. To this end, we developed a strategy for high-yield cortisone production based on ectopic expression of the guinea-pig 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in Bacillus megaterium. We screened different constructs, containing a variety of promoters tailored for B. megaterium, and created modified versions of the enzyme by protein engineering to optimize cortisone yield. Furthermore, we utilized co-expression of an alcohol dehydrogenase to promote NADP+ regeneration, which significantly improved 11ß-HSD1 activity. The process thereby developed was found to show a remarkably high regioselectivity of >95% and to generate cortisone yields of up to 13.65 g L-1 d-1, which represents a ∼1000-fold improvement over the next-best reported system. In summary, we demonstrate the utility of B. megaterium MS941 as a suitable host for recombinant protein production and its high potential for industrial steroid production.

A novel short chain dehydrogenase from Bacillus megaterium for the conversion of the sesquiterpene nootkatol to (+)-nootkatone.

(+)-Nootkatone is a natural ingredient that occurs in grapefruit and certain other plants and is responsible for the characteristic smell of grapefruit. Due to its versatile applications in the flavor and fragrance industry as well as its application in some medical uses it recruits the interests of academic research along with industrial biotechnology. In the current work we present the application of a novel short chain dehydrogenase from Bacillus megaterium in an in vivo whole-cell biocatalyst system for the conversion of the intermediate nootkatol into the industrially valuable (+)-nootkatone. The newly identified dehydrogenase converted nootkatol selectively and efficiently into the final product. The conversion ratio of about 100% was achieved within 40 min yielding about 44 mg/L (+)-nootkatone. Furthermore, the herein identified dehydrogenase provides a new tool to overcome the limitation of the two-step enzymatic biotechnological process for the production of (+)-nootkatone.

Expanding the promoter toolbox of Bacillus megaterium.

Over the past decades, Bacillus megaterium has gained significant interest in the biotechnological industry due to its high capacity for protein production. Although many proteins have been expressed efficiently using the optimized xylose inducible system so far, there is a considerable demand for novel promoters with varying activities, particularly for the adjustment of protein levels in multi-enzyme cascades. Genome-wide microarray analyses of the industrially important B. megaterium strain MS941 were applied to identify constitutive and growth phase dependent promoters for the expression of heterologous proteins from the early exponential to the early stationary phase of bacterial growth. Fifteen putative promoter elements were selected based on differential gene expression profiles and signal intensities of the generated microarray data. The corresponding promoter activities were evaluated in B. megaterium via ß-galactosidase screening. ß-Galactosidase expression levels ranged from 15% to 130% compared to the optimized xylose inducible promoter. Apart from these constitutive promoters we also identified and characterized novel inducible promoters, which were regulated by the addition of arabinose, galactose and the commonly used allolactose analog IPTG. The potential application of the identified promoters for biotechnologically relevant processes was demonstrated by overexpression of the cholesterol oxidase II from Brevibacterium sterolicum, thus obtaining product yields of up to 1.13 g/l/d. The provided toolbox of novel promoters offers versatile promoter strengths and will significantly contribute to harmonize protein expression in synthetic metabolic pathways, thereby pushing forward the engineering of B. megaterium as microbial cell factory for the biosynthesis and conversion of valuable compounds.

Characterization and engineering of a carotenoid biosynthesis operon from Bacillus megaterium.

Bacillus megaterium belongs to the group of pigmented bacilli producing carotenoids that ensure self-protection from UV radiation-induced and collateral oxidative damage. Metabolite profiling of strain MS941 revealed the presence of the C30 carotenoids 4,4'-diapophytofluene and 4,4'-diaponeurosporenic acid. A gene function analysis demonstrated the presence of a corresponding C30 carotenoid biosynthetic pathway with pharmaceutical importance. We identified a gene cluster comprising putative genes for a farnesyl diphosphate synthase (IspA), a diapophytoene synthase (CrtM) and three distinct diapophytoene desaturases (CrtN1-3). Intriguingly, crtM was organized in an operon together with two of the identified crtN genes. The individual activities of the encoded enzymes were determined by heterologous expression and product analysis in the non-carotenogenic model organism Escherichia coli. Our experimental data show that the first catalytic steps of C30 carotenoid biosynthesis in B. megaterium share significant similarity to the corresponding biosynthetic pathway of Staphylococcus aureus. The biosynthesis of farnesyl diphosphates and their subsequent condensation to form 4,4'-diapophytoene are catalyzed by the identified IspA and CrtM, respectively. The following desaturation reactions to form 4,4'-diaponeurosporene, however, require the activities of multiple diapophytoene desaturases. A biosynthetic operon was engineered and successfully expressed in an E. coli whole-cell system creating a cell factory for a high-yield production of the C30 carotenoid 4,4'-diaponeurosporene which has promising potential in the treatment of various inflammatory diseases.

Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin.

In the current work, we describe the identification and characterization of the first plasmid-encoded P450 (CYP107DY1) from a Bacillus species. The recombinant CYP107DY1 exhibits characteristic P450 absolute and reduced CO-bound difference spectra. Reconstitution with different redox systems revealed the autologous one, consisting of BmCPR and Fdx2, as the most effective one. Screening of a library of 18 pharmaceutically relevant compounds displayed activity towards mevastatin to produce pravastatin. Pravastatin is an important therapeutic drug to treat hypercholesterolemia, which was described to be produced by oxyfunctionlization of mevastatin (compactin) by members of CYP105 family. The hydroxylation at C6 of mevastatin was also suggested by docking this compound into a computer model created for CYP107DY1. Moreover, in view of the biotechnological application, CYP107DY1 as well as its redox partners (BmCPR and Fdx2) were successfully utilized to establish an E. coli based whole-cell system for an efficient biotransformation of mevastatin. The in vitro and in vivo application of the CYP07DY1 also offers the possibility for the screening of more substrates, which could open up further biotechnological usage of this enzyme.

Genetic engineering of Bacillus megaterium for high-yield production of the major teleost progestogens 17α,20ß-di- and 17α,20ß,21α-trihydroxy-4-pregnen-3-one.

17α,20ß-Dihydroxy-4-pregnen-3-one (17α,20ßDiOH-P) and 17α,20ß,21α-trihydroxy-4-pregnen-3-one (20ßOH-RSS) are the critical hormones required for oocyte maturation in fish. We utilized B. megaterium's endogenous 20ß-hydroxysteroid dehydrogenase (20ßHSD) for the efficient production of both progestogens after genetically modifying the microorganism to reduce side-product formation. First, the gene encoding the autologous cytochrome P450 CYP106A1 was deleted, resulting in a strain devoid of any steroid hydroxylation activity. Cultivation of this strain in the presence of 17α-hydroxyprogesterone (17αOH-P) led to the formation of 17α,20α-dihydroxy-4-pregnen-3-one (17α,20αDiOH-P) as a major and 17α,20ßDiOH-P as a minor product. Four enzymes were identified as 20αHSDs and their genes deleted to yield a strain with no 20αHSD activity. The 3-oxoacyl-(acyl-carrier-protein) reductase FabG was found to exhibit 20ßHSD-activity and overexpressed to create a biocatalyst yielding 0.22g/L 17α,20ßDiOH-P and 0.34g/L 20ßOH-RSS after 8h using shake-flask cultivation, thus obtaining products that are at least a thousand times more expensive than their substrates.

Occurrence of rheumatic carditis in the native population of Curacao, Netherlands West Indies

Among 3,391 admissions for internal diseases at Curacao, Netherlands West Indies, over a period of five years, there were 61 for acute rheumatic fever. Three cases were complicated by chorea minor. Scarlet fever was not observed. Among 1,307 autopsies on native of Curacao, Aruba and Bonaire there were 20 which disclosed typical gross lesions or sequelae of rheumatic carditis. In 12 of these histologic examination was possible, and in 11 typical Aschoff bodies were found. The supposition is advanced that rheumatic carditis is more frequent in the tropics than is commonly believed but that the true incidence can be determined only by collecting reliable data, based especially on autopsies with histologic examinations. (summary)