Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase.

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement-a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound-and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.

The Development and Opportunities of Predictive Biotechnology.

Recent advances in bioeconomy allow a holistic view of existing and new process chains and enable novel production routines continuously advanced by academia and industry. All this progress benefits from a growing number of prediction tools that have found their way into the field. For example, automated genome annotations, tools for building model structures of proteins, and structural protein prediction methods such as AlphaFold2TM or RoseTTAFold have gained popularity in recent years. Recently, it has become apparent that more and more AI-based tools are being developed and used for biocatalysis and biotechnology. This is an excellent opportunity for academia and industry to accelerate advancements in the field further. Biotechnology, as a rapidly growing interdisciplinary field, stands to benefit greatly from these developments.

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris.

Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol-3-phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC-like activity was supported by the functional complementation of a temperature-sensitive plsC-deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC-like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent-solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes.

Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives.

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

Structural and Mechanistic Studies on Substrate and Stereoselectivity of the Indole Monooxygenase VpIndA1: New Avenues for Biocatalytic Epoxidations and Sulfoxidations.

Flavoprotein monooxygenases are a versatile group of enzymes for biocatalytic transformations. Among these, group E monooxygenases (GEMs) catalyze enantioselective epoxidation and sulfoxidation reactions. Here, we describe the crystal structure of an indole monooxygenase from the bacterium Variovorax paradoxus EPS, a GEM designated as VpIndA1. Complex structures with substrates reveal productive binding modes that, in conjunction with force-field calculations and rapid mixing kinetics, reveal the structural basis of substrate and stereoselectivity. Structure-based redesign of the substrate cavity yielded variants with new substrate selectivity (for sulfoxidation of benzyl phenyl sulfide) or with greatly enhanced stereoselectivity (from 35.1 % to 99.8 % ee for production of (1S,2R)-indene oxide). This first determination of the substrate binding mode of GEMs combined with structure-function relationships opens the door for structure-based design of these powerful biocatalysts.

Cell-Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor.

Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications.

Microbial Degradation of Azo Dyes: Approaches and Prospects for a Hazard-Free Conversion by Microorganisms.

Azo dyes have become a staple in various industries, as colors play an important role in consumer choices. However, these dyes pose various health and environmental risks. Although different wastewater treatments are available, the search for more eco-friendly options persists. Bioremediation utilizing microorganisms has been of great interest to researchers and industries, as the transition toward greener solutions has become more in demand through the years. This review tackles the health and environmental repercussions of azo dyes and its metabolites, available biological approaches to eliminate such dyes from the environment with a focus on the use of different microorganisms, enzymes that are involved in the degradation of azo dyes, and recent trends that could be applied for the treatment of azo dyes.

Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase.

Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone - giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).

In vitro and in silico analysis of Brilliant Black degradation by Actinobacteria and a Paraburkholderia sp.

The soil bacteria isolated in this study, including three strains of actinobacteria and one Paraburkholderia sp., showed decolorization activity of azo dyes in the resting cell assay and were shown to use methyl red as the sole carbon source to proliferate. Therefore, their ability to degrade, bioabsorb, or a combination of both mechanism was investigated using the substrate brilliant black. The strains DP-A9 and DP-L11, within 24 h of incubation, showed complete biodegradation of 173.54 mg/L brilliant black and the strains DP-D10 and DP-P12 showed partial decolorization of 83.3 mg/L and 36.4 mg/L, respectively, by both biosorption and biodegradation. In addition, the shotgun assembled genome of these strains showed a highly diverse set of genes encoding for candidate dye degrading enzymes, providing avenues to study azo dye metabolism in more detail.

Improving Biocatalytic Properties of an Azoreductase via the N-Terminal Fusion of Formate Dehydrogenase.

Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)+ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDHC23S and FDHC23SD195QY196H ) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDHC23S +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDHC23S +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.

Correction to: Draft genome sequence of Kocuria indica DP-K7, a methyl red degrading actinobacterium.

[This corrects the article DOI: 10.1007/s13205-020-2136-3.].

Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade.

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative ß-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases.

Secondary metabolites released by the rhizosphere bacteria Arthrobacter oxydans and Kocuria rosea enhance plant availability and soil-plant transfer of germanium (Ge) and rare earth elements (REEs).

Here, we explore effects of metallophore-producing rhizobacteria on the plant availability of germanium (Ge) and rare earth elements (REEs). Five isolates of the four species Rhodococcus erythropolis, Arthrobacter oxydans, Kocuria rosea and Chryseobacterium koreense were characterized regarding their production of element-chelators using genome-mining, LC-MS/MS analysis and solid CAS-assay. Additionally, a soil elution experiment was conducted in order to identify isolates that increase solubility of Ge and REEs in soil solution. A. oxydans ATW2 and K. rosea ATW4 released desferrioxamine-, bacillibactin- and surfactin-like compounds that mobilized Ge and REEs as well as P, Fe, Si and Ca in soil. Subsequently, oat, rapeseed and reed canary grass were cultivated on soil and sand and treated with cells and iron depleted culture supernatants of A. oxydans ATW2 and K. rosea ATW4. Inoculation increased plant yield and shoot phosphorus (P), manganese (Mn), Ge and REE concentrations. However, effects of the inoculation varied substantially between the growth substrates and plant species. On sand, A. oxydans ATW2 increased accumulation of REEs in all plant species and root-shoot translocation in rapeseed, while K. rosea ATW4 enhanced REE accumulation in rapeseed only, without effects on other plant species. Sand-cultured oat plants showed increased Ge accumulation and root-shoot translocation in presence of A. oxydans ATW2 cells and K. rosea ATW4 supernatant; however, there was no effect on other plant species, irrespective the growth substrate used. In contrast, soil-cultured rapeseed showed enhanced REE accumulation in presence of cells of A. oxydans ATW2 while there were no effects on other plant species and Ge. The processes involved are not yet fully understood. Nevertheless, we demonstrated that chemical microbe-soil-plant relationships influence plant availability of nutrients together with Ge and REEs, which has major implications on our understanding of biogeochemical element cycling and development of sustainable bioremediation and biomining technologies.

Characterization of the Glutathione S-Transferases Involved in Styrene Degradation in Gordonia rubripertincta CWB2.

The glutathione S-transferases carried on the plasmid for the styrene-specific degradation pathway in the Actinobacterium Gordonia rubripertincta CWB2 were heterologously expressed in Escherichia coli. Both enzymes were purified via affinity chromatography and subjected to activity investigations. StyI and StyJ displayed activity toward the commonly used glutathione S-transferase model substrate 1-chloro-2,4-dinitrobenzene (CDNB) with Km values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and Vmax values of 0.0158 ± 0.0002 and 0.348 ± 0.008 U mg-1 for StyI and StyJ, respectively. The conversion of the natural substrate styrene oxide to the intermediate (1-phenyl-2-hydroxyethyl)glutathione was detected for StyI with 48.3 ± 2.9 U mg-1. This elucidates one more step in the not yet fully resolved styrene-specific degradation pathway of Gordonia rubripertincta CWB2. A characterization of both purified enzymes adds more insight into the scarce research field of actinobacterial glutathione S-transferases. Moreover, a sequence and phylogenetic analysis puts both enzymes into a physiological and evolutionary context. IMPORTANCE Styrene is a toxic compound that is used at a large scale by industry for plastic production. Bacterial degradation of styrene is a possibility for bioremediation and pollution prevention. Intermediates of styrene derivatives degraded in the styrene-specific pathways are precursors for valuable chemical compounds. The pathway in Gordonia rubripertincta CWB2 has proven to accept a broader substrate range than other bacterial styrene degraders. The enzymes characterized in this study, distinguish CWB2s pathway from other known styrene degradation routes and thus might be the main key for its ability to produce ibuprofen from the respective styrene derivative. A biotechnological utilization of this cascade could lead to efficient and sustainable production of drugs, flavors, and fragrances. Moreover, research on glutathione metabolism in Actinobacteria is rare. Here, a characterization of two glutathione S-transferases of actinobacterial origin is presented, and the utilization of glutathione in the metabolism of an Actinobacterium is proven.

Natural diversity of FAD-dependent 4-hydroxybenzoate hydroxylases.

4-Hydroxybenzoate 3-hydroxylase (PHBH) is the most extensively studied group A flavoprotein monooxygenase (FPMO). PHBH is almost exclusively found in prokaryotes, where its induction, usually as a consequence of lignin degradation, results in the regioselective formation of protocatechuate, one of the central intermediates in the global carbon cycle. In this contribution we introduce several less known FAD-dependent 4-hydroxybenzoate hydroxylases. Phylogenetic analysis showed that the enzymes discussed here reside in distinct clades of the group A FPMO family, indicating their separate divergence from a common ancestor. Protein homology modelling revealed that the fungal 4-hydroxybenzoate 3-hydroxylase PhhA is structurally related to phenol hydroxylase (PHHY) and 3-hydroxybenzoate 4-hydroxylase (3HB4H). 4-Hydroxybenzoate 1-hydroxylase (4HB1H) from yeast catalyzes an oxidative decarboxylation reaction and is structurally similar to 3-hydroxybenzoate 6-hydroxylase (3HB6H), salicylate hydroxylase (SALH) and 6-hydroxynicotinate 3-monooxygenase (6HNMO). Genome mining suggests that the 4HB1H activity is widespread in the fungal kingdom and might be responsible for the oxidative decarboxylation of vanillate, an import intermediate in lignin degradation. 4-Hydroxybenzoyl-CoA 1-hydroxylase (PhgA) catalyzes an intramolecular migration reaction (NIH shift) during the three-step conversion of 4-hydroxybenzoate to gentisate in certain Bacillus species. PhgA is phylogenetically related to 4-hydroxyphenylacetate 1-hydroxylase (4HPA1H). In summary, this paper shines light on the natural diversity of group A FPMOs that are involved in the aerobic microbial catabolism of 4-hydroxybenzoate.

Flavoprotein monooxygenases: Versatile biocatalysts.

Flavoprotein monooxygenases (FPMOs) are single- or two-component enzymes that catalyze a diverse set of chemo-, regio- and enantioselective oxyfunctionalization reactions. In this review, we describe how FPMOs have evolved from model enzymes in mechanistic flavoprotein research to biotechnologically relevant catalysts that can be applied for the sustainable production of valuable chemicals. After a historical account of the development of the FPMO field, we explain the FPMO classification system, which is primarily based on protein structural properties and electron donor specificities. We then summarize the most appealing reactions catalyzed by each group with a focus on the different types of oxygenation chemistries. Wherever relevant, we report engineering strategies that have been used to improve the robustness and applicability of FPMOs.

Isolation and characterization of arsenic-binding siderophores from Rhodococcus erythropolis S43: role of heterobactin B and other heterobactin variants.

Rhodococcus erythropolis S43 is an arsenic-tolerant actinobacterium isolated from an arsenic contaminated soil. It has been shown to produce siderophores when exposed to iron-depleting conditions. In this work, strain S43 was shown to have the putative heterobactin production cluster htbABCDEFGHIJ(K). To induce siderophore production, the strain was cultured in iron-depleted medium in presence and absence of sodium arsenite. The metabolites produced by S43 in the colorimetric CAS and As-mCAS assays, respectively, showed iron- and arsenic-binding properties reaching a chelating activity equivalent to 1.6 mM of desferroxamine B in the supernatant of the culture without arsenite. By solid-phase extraction and two subsequent HPLC separations from both cultures, several fractions were obtained, which contained CAS and As-mCAS activity and which were submitted to LC-MS analyses including fragmentation of the major peaks. The mixed-type siderophore heterobactin B occurred in all analyzed fractions, and the mass of the "Carrano heterobactin A" was detected as well. In addition, generation of a molecular network based on fragment spectra revealed the occurrence of several other compounds with heterobactin-like structures, among them a heterobactin B variant with an additional CH2O moiety. 1H NMR analyses obtained for preparations from the first HPLC step showed signals of heterobactin B and of "Carrano heterobactin A" with different relative amounts in all three samples. In summary, our results reveal that in R. erythropolis S43, a pool of heterobactin variants is responsible for the iron- and arsenic-binding activities. KEY POINTS: • Several heterobactin variants are the arsenic-binding compounds in Rhodococcus erythropolis S43. • Heterobactin B and the compound designated heterobactin A by Carrano are of importance. • In addition, other heterobactins with ornithine in the backbone exist, e.g., the new heterobactin C.

Screening for Microbial Metal-Chelating Siderophores for the Removal of Metal Ions from Solutions.

To guarantee the supply of critical elements in the future, the development of new technologies is essential. Siderophores have high potential in the recovery and recycling of valuable metals due to their metal-chelating properties. Using the Chrome azurol S assay, 75 bacterial strains were screened to obtain a high-yield siderophore with the ability to complex valuable critical metal ions. The siderophore production of the four selected strains Nocardioides simplex 3E, Pseudomonas chlororaphis DSM 50083, Variovorax paradoxus EPS, and Rhodococcus erythropolis B7g was optimized, resulting in significantly increased siderophore production of N. simplex and R. erythropolis. Produced siderophore amounts and velocities were highly dependent on the carbon source. The genomes of N. simplex and P. chlororaphis were sequenced. Bioinformatical analyses revealed the occurrence of an achromobactin and a pyoverdine gene cluster in P. chlororaphis, a heterobactin and a requichelin gene cluster in R. erythropolis, and a desferrioxamine gene cluster in N. simplex. Finally, the results of the previous metal-binding screening were validated by a proof-of-concept development for the recovery of metal ions from aqueous solutions utilizing C18 columns functionalized with siderophores. We demonstrated the recovery of the critical metal ions V(III), Ga(III), and In(III) from mixed metal solutions with immobilized siderophores of N. simplex and R. erythropolis.

Biochemical Characterization of Phenylacetaldehyde Dehydrogenases from Styrene-degrading Soil Bacteria.

Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.