Characterization of Fungal FAD-Dependent AA3_2 Glucose Oxidoreductases from Hitherto Unexplored Phylogenetic Clades

The CAZy auxiliary activity family 3 (AA3) comprises FAD-dependent enzymes belonging to the superfamily of glucose-methanol-choline (GMC) oxidoreductases. Glucose oxidase (GOx; EC 1.1.3.4) and glucose dehydrogenase (GDH; EC 1.1.5.9) are part of subfamily AA3_2 and catalyze the oxidation of β-D-glucose at its anomeric carbon to D-glucono-1,5-lactone. Recent phylogenetic analysis showed that AA3_2 glucose oxidoreductases can be grouped into four major clades, GOx I and GDH I–III, and in minor clades such as GOx II or distinct subclades. This wide sequence space of AA3_2 glucose oxidoreductases has, however, not been studied in detail, with mainly members of GOx I and GDH I studied biochemically or structurally. Here, we report the biochemical characterization of four fungal glucose oxidoreductases from distinct, hitherto unexplored clades or subclades. The enzyme from Aureobasidium subglaciale, belonging to the minor GOx II clade, showed a typical preference for oxygen and glucose, confirming the correct annotation of this clade. The other three enzymes exhibited strict dehydrogenase activity with different substrate specificities. GDH II from Trichoderma virens showed an almost six-fold higher catalytic efficiency for maltose compared to glucose. The preferred substrate for the two GDH III enzymes from Rhizoctonia solani and Ustilago maydis was gentiobiose, a β(1→6) disaccharide, as judged from the catalytic efficiency. Overall, the newly studied AA3_2 glucose oxidoreductases showed a much broader substrate spectrum than the archetypal GOx from Aspergillus niger, which belongs to clade GOx I.

Arylmalonate Decarboxylase—A Versatile Biocatalyst for the Synthesis of Optically Pure Carboxylic Acids

Bacterial arylmalonate decarboxylase (AMDase) is an intriguing cofactor-independent enzyme with a broad substrate spectrum. Particularly, the highly stereoselective transformation of diverse arylmalonic acids into the corresponding chiral α-arylpropionates has contributed to the broad recognition of this biocatalyst. While, more than 30 years after its discovery, the native substrate and function of AMDase still remain undiscovered, contributions from multiple fields have ever since brought forth a powerful collection of AMDase variants to access a wide variety of optically pure α-substituted propionates. This review aims at providing a comprehensive overview of the development of AMDase from an enzyme with unknown function up to a powerful tailored biocatalyst for the synthesis of industrially relevant optically pure α-arylpropionates. Historical perspectives as well as recent achievements in the field will be covered within this work.

Cloning, expression, and characterization of Baeyer–Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer–Villiger oxidation of 4–10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in Escherichia coli and its substrate spectrum and kinetic parameters investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer–Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4–C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer–Villiger monooxygenases were also cloned and expressed in Escherichia coli.

Transport of Drugs and Endogenous Compounds Mediated by Human OCT1: Studies in Single- and Double-Transfected Cell Models

Organic Cation Transporter 1 (OCT1, gene symbol: SLC22A1) is predominately expressed in human liver, localized in the basolateral membrane of hepatocytes and facilitates the uptake of endogenous compounds (e.g. serotonin, acetylcholine, thiamine), and widely prescribed drugs (e.g. metformin, fenoterol, morphine). Furthermore, exogenous compounds such as MPP+, ASP+ and Tetraethylammonium can be used as prototypic substrates to study the OCT1-mediated transport in vitro. Single-transfected cell lines recombinantly overexpressing OCT1 (e.g., HEK-OCT1) were established to study OCT1-mediated uptake and to evaluate transporter-mediated drug-drug interactions in vitro. Furthermore, double-transfected cell models simultaneously overexpressing basolaterally localized OCT1 together with an apically localized export protein have been established. Most of these cell models are based on polarized grown MDCK cells and can be used to analyze transcellular transport, mimicking the transport processes e.g. during the hepatobiliary elimination of drugs. Multidrug and toxin extrusion protein 1 (MATE1, gene symbol: SLC47A1) and the ATP-driven efflux pump P-glycoprotein (P-gp, gene symbol: ABCB1) are both expressed in the canalicular membrane of human hepatocytes and are described as transporters of organic cations. OCT1 and MATE1 have an overlapping substrate spectrum, indicating an important interplay of both transport proteins during the hepatobiliary elimination of drugs. Due to the important role of OCT1 for the transport of endogenous compounds and drugs, in vitro cell systems are important for the determination of the substrate spectrum of OCT1, the understanding of the molecular mechanisms of polarized transport, and the investigation of potential drug-drug interactions. Therefore, the aim of this review article is to summarize the current knowledge on cell systems recombinantly overexpressing human OCT1.

The assessment of microbiological diagnostics’ credibility in Poland on the basis of the POLMICRO 2019 Program results

Introduction: This article describes the course and results of the 26th edition of the Polish National External Quality Assessment Scheme in Microbiology (POLMICRO). Aim: The aim is to assess the credibility of diagnostics conducted by microbiological laboratories in Poland, as well as the results and organization of the POLMICRO/ MIKOLOGIA (MYCOLOGY) and POLMICRO/SSE programs. Material and methods: The POLMICRO 2019 edition, according to the schedule, consisted of six rounds: five practical rounds and one theoretical round of an educational nature. The number of POLMICRO 2019 participants varied, depending on the round, from 430 to 438. In total, 443 laboratories took part in the program. The most (n = 438) laboratories took part in round I, and the least (n = 430) in round VI. Results: The shape and color of all test objects in the round devoted to microscopic preparations (4th round of POLMICRO 2019) were correctly assessed by 97.0% of laboratories (n = 419). Gram-negative rod strains belonging to the species Escherichia coli (PM-366, PM-367) and Klebsiella pneumoniae (PM-368, PM-369), from infection of the blood bed without a palpable site of infection, which were the subjects of the second round of POLMICRO 2019 studies, all participants correctly identified (100%; n = 433). While in the case of the MBL (metallo-β-lactamase) mechanism all participants (n = 439) correctly interpreted the presented picture, in the case of ESBL (β-lactamase with an extended substrate spectrum), seven laboratories (1.6%) misinterpreted the attached photos of phenotypic tests. Conclusions: In 2019, the Central Center for Quality Research in Microbiological Diagnostics, as previously planned, expanded its activity in the field of POLMICRO external laboratory control to include programs for mycological diagnostics (POLMICRO/ MIKOLOGIA) and diagnostics of etiological factors of gastrointestinal infections (POLMICRO/ SSE). Both the plan and the educational goals of the program have been achieved.

Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

Structural and functional insights into esterase-mediated macrolide resistance

Macrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides’ macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.

Study on the Activity of a New Amidase Signature (AS) Family

This study cloned a new amidase signature (AS) family gene from Escherichia coli (E. coii), and mainly investigated the activity of this enzyme. The protein, according to the bioinformatics analysis, belonged to the AS family. It was expressed, purified and characterized in E. coli BL21 (DE3). As a result, 40 °C and pH 8.0 were the optimum temperature and pH. It showed a wide substrate spectrum and high activity for aromatic and aliphatic amides, while with a narrow range of anilide substrate, and only propanol could be adopted as an effective substrate. Because of its wide range of substrate specificities, this will boost the applicability of amidase and make it have the potential for a wide range of applications in biosynthesis and biodegradation.

Characterization of different biocatalyst formats for BVMO-catalyzed cyclohexanone oxidation

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Further, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, i.e., isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for K (9.3-fold higher) and k (4.8-fold lower) compared to corresponding K and k values of isolated CHMO, but a favorable K for cyclohexanone (5.3-fold higher). The unfavorable K and k values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, in order to exploit the high potential of biofilms regarding process stability. Suspended cells showed an only 1.8-fold higher K, but 1.3- and 4.2-fold higher k and K values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

A new clade of styrene monooxygenases for (R)-selective epoxidation

Styrene monooxygenases (SMOs) are excellent enzymes for the production of (S)-enantiopure epoxides, but so far, only one (R)-selective SMO has been identified with a narrow substrate spectrum.