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Special features of the protopectin complex structure of plant tissue suggest the necessity of performing point destruction of certain glycoside bonds in the structure of rhamnogalacturonan polymer chains for industrial production of pectin. These chains include homogalacturonan sites and branching zones. As the homogalacturonan fragments of the protopectin complex carry the main functional load, glycoside bonds between residues of rhamnose and galacturonic acid are targeted bonds. For their directional destruction, it is most expedient to use enzymes of lyase and hydrolase action. The aim of this review is to systemize notions of molecular specific features of enzymes of lyase and hydrolase action that catalyze the process of enzymatic destruction of the rhamnogalacturonan main chain. The paper examines systematics of lyase and hydrolase enzymes by mechanism of destruction of glycoside bonds and by molecular structure. It is shown that the classification data intercross, as a result, each family can include one or several enzyme groups. The review shows the main structural difference of enzymes of lyase and hydrolase action that consists in the obligatory presence of Ca2+ cations in the composition of lyase enzymes. These cations take part in stabilization of conformation of the enzyme molecule and in the catalytic process per se blocking the residue of galacturonic acid. Ca2+ cations are absent in the composition of targeted hydrolase enzymes. Molecular specific features of lyase enzymes determine sensitivity of their catalytic activity to the presence of Ca2+ cations in the system. Exceeding certain concentration can lead to the antagonistic effect. There is no unambiguous idea of this regarding hydrolase enzymes. The review demonstrates the necessity of studying approaches to assessment of expediency of preliminary partial removal of cations from the substrate.

Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya, Leptothermofonsia, Ocullathermofonsia, Thermoleptolyngbya, Trichothermofonsia, Synechococcus, Thermostichus, and Thermosynechococcus. According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE, cpcF, and cpcT. In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement. [ABSTRACT FROM AUTHOR]

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy- l - erythro -hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α- l -guluronate oligosaccharides with a lower content of β- d -mannuronate residues compared to what can be achieved using chemical depolymerization alone. [Display omitted] [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Bioprocess Engineering is the property of Chinese Journal of Bioprocess Engineering Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Self-assembling enzyme mimics offer an easy way to imitate activities of natural enzymes but have not been thus far used to understand the effect of different amino acids on the catalytic activity and why they are evolutionarily preserved for specific catalytic roles. Here, we demonstrated that fullerene nanostructures functionalized with catalytically active amino acids, which form multiple active sites via the self-assembly process in the aqueous environment, serve as an effective system to distinguish the catalytic activity differences resulting from single amino acid changes. A nano-level tuning of intermolecular and intramolecular interactions enabled formation of efficient enzyme mimics. Furthermore, using the carboxyl–imidazole couple found in quite different enzymes as the main catalytic unit, we could mimic different enzyme classes, like hydrolases and lyases, with significant catalytic activities. These designed nanocatalysts were also reusable and catalytically active under physiological conditions like natural enzymes. [ABSTRACT FROM AUTHOR]

In this study, we aimed to isolate bacteria capable of degrading the polysaccharide ulvan from the green algae Ulva sp. (Chlorophyta, Ulvales, Ulvaceae) in marine environments. We isolated 13 ulvan-degrading bacteria and observed high diversity at the genus level. Further, the genera Paraglaciecola, Vibrio, Echinicola, and Algibacter, which can degrade ulvan, were successfully isolated for the first time from marine environments. Among the 13 isolates, only one isolate (Echinicola sp.) showed the ability not only to produce externally expressed ulvan lyase, but also to be periplasmic or on the cell surface. From the results of the full-genome analysis, lyase was presumed to be a member of the PL25 (BNR4) family of ulvan lyases, and the bacterium also contained the sequence for glycoside hydrolase (GH43, GH78 and GH88), which is characteristic of other ulvan-degrading bacteria. Notably, this bacterium has a unique ulvan lyase gene not previously reported. [ABSTRACT FROM AUTHOR]

Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.

The Streptococcus mutans (S. mutans) phage, as one of the principal pathogenic bacteria of dental caries, is a main cause of the formation and development of dental caries due to its overproliferation in dental plaque biofilms. Bacterial viruses, also known as bacteriophages, have the capability of specifically infecting bacteria and effectively degrading bacterial biofilms. S. mutans phages, therefore, may prevent and control caries. Therapy based on phages has been applied in many fields, but the application of S. mutans phages in caries remains exploratory. This article will review the research progress of S. mutans phages in clinical caries prevention, aiming to provide a new idea for the clinical prevention of caries. The results of the literature review show that the living bacteriophage system has the advantages of high specificity, high affinity and good safety. However, due to its unstable structure, it can be processed into a more stable formulation by freeze-drying, spray drying, adding stability enhancers, or incorporating bacteriophages into ointments, biodegradable polymer matrices or particles to a certain extent to improve stability. The lysozyme produced by phages can digest the bacterial cell wall and release the assembled phage particles, which effectively cleave biofilms. In addition, the antigen binding fragment library for cariogenic pathogens was screened by phage display technology, and the purpose of caries prevention and treatment was achieved by passive immunization of antigen binding fragments. However, the host range of bacteriophages is narrow, so this kind of problem can be overcome by phage combined with traditional therapy or other drug use or cocktail therapy with multiple phages in clinical caries prevention and control.

Thermophilic cyanobacteria are cosmopolitan and abundant in the thermal environment. Their light-harvesting complexes, phycobilisomes (PBS), are highly important in photosynthesis. To date, there is limited information on the PBS composition of thermophilic cyanobacteria whose habitats are challenging for survival. Herein, genome-based methods were used to investigate the molecular components of PBS in 19 well-described thermophilic cyanobacteria. These cyanobacteria are from the genera Leptolyngbya, Leptothermofonsia, Ocullathermofonsia, Thermoleptolyngbya, Trichothermofonsia, Synechococcus, Thermostichus, and Thermosynechococcus. According to the phycobiliprotein (PBP) composition of the rods, two pigment types are observed in these thermophiles. The amino acid sequence analysis of different PBP subunits suggests several highly conserved cysteine residues in these thermophiles. Certain amino acid contents in the PBP of thermophiles are significantly higher than their mesophilic counterparts, highlighting the potential roles of specific substitutions of amino acid in the adaptive thermostability of light-harvesting complexes in thermophilic cyanobacteria. Genes encoding PBS linker polypeptides vary among the thermophiles. Intriguingly, motifs in linker apcE indicate a photoacclimation of a far-red light by Leptolyngbya JSC-1, Leptothermofonsia E412, and Ocullathermofonsia A174. The composition pattern of phycobilin lyases is consistent among the thermophiles, except for Thermostichus strains that have extra homologs of cpcE, cpcF, and cpcT. In addition, phylogenetic analyses of genes coding for PBPs, linkers, and lyases suggest extensive genetic diversity among these thermophiles, which is further discussed with the domain analyses. Moreover, comparative genomic analysis suggests different genomic distributions of PBS-related genes among the thermophiles, indicating probably various regulations of expression. In summary, the comparative analysis elucidates distinct molecular components and organization of PBS in thermophilic cyanobacteria. These results provide insights into the PBS components of thermophilic cyanobacteria and fundamental knowledge for future research regarding structures, functions, and photosynthetic improvement.

Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Sialo‐oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio‐catalysis is central to the development of sialo‐oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialo‐oligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling‐guided approach to 3ʹ‐sialyllactose (3SL) synthesis from N‐acetyl‐ d‐neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ‐triphosphate, via the reactions of cytidine 5ʹ‐monophosphate‐Neu5Ac synthetase and α2,3‐sialyltransferase. The Neu5Ac was synthesized in situ from N‐acetyl‐ d‐mannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase. We show through comprehensive time‐course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model‐based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%–75%; 7–10 g/L) and overall productivity (3–5 g/L/h). Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme‐catalyzed transformations fit for oligosaccharide production. [ABSTRACT FROM AUTHOR]

Main observation and conclusion: The radical S‐adenosylmethionine (SAM) enzyme NosL catalyzes the conversion of L‐tryptophan (L‐Trp, 1) to 3‐methyl‐2‐indolic acid (MIA, 2), a key intermediate in the biosynthesis of the peptide antibiotic nosiheptide. Previous study showed that this remarkable recombination reaction starts from the cleavage of the Cα—COO– bond to result in a •CO2− radical migration. In contrast to the radical SAM tyrosine lyases, NosL appears unable to cleave the Cα—Cβ bond, which is intrinsically more favorable to be cleaved than the Cα—COO– bond. In this study, we investigate the NosL activity with tryptamine (11) and tryptophol (12), two L‐Trp analogues lacking a carboxylate moiety. We showed that NosL cleaves the C1—C2 bond of these two substrates to produce 3‐methylindole (7), suggesting that the enzyme can still catalyze a β‐scission when the carboxyl group of Trp is absent. We also showed the enzyme exhibits a promiscuous activity, initiating the reaction by abstracting hydrogen atoms from two different sites to produce two sets of products. [ABSTRACT FROM AUTHOR]

The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

In this study, we aimed to isolate bacteria capable of degrading the polysaccharide ulvan from the green algae Ulva sp. (Chlorophyta, Ulvales, Ulvaceae) in marine environments. We isolated 13 ulvan-degrading bacteria and observed high diversity at the genus level. Further, the genera Paraglaciecola, Vibrio, Echinicola, and Algibacter, which can degrade ulvan, were successfully isolated for the first time from marine environments. Among the 13 isolates, only one isolate (Echinicola sp.) showed the ability not only to produce externally expressed ulvan lyase, but also to be periplasmic or on the cell surface. From the results of the full-genome analysis, lyase was presumed to be a member of the PL25 (BNR4) family of ulvan lyases, and the bacterium also contained the sequence for glycoside hydrolase (GH43, GH78 and GH88), which is characteristic of other ulvan-degrading bacteria. Notably, this bacterium has a unique ulvan lyase gene not previously reported.

Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems.

Ulvan is a complex sulfated polysaccharide extracted from Ulva , and ulvan lyases can degrade ulvan through a β-elimination mechanism to obtain oligosaccharides. In this study, a new ulvan lyase, EPL15085, which belongs to the polysaccharide lyase (PL) 28 family from Tamlana fucoidanivorans CW2-9, was characterized in detail. The optimal pH and salinity are 9.0 and 0.4 M NaCl, respectively. The K m and V max of recombinant EPL15085 toward ulvan are 0.80 mg·mL -1 and 11.22 μmol·min -1 mg -1 ·mL -1 , respectively. Unexpectedly, it is very resistant to high temperatures. After treatment at 100 °C, EPL15085 maintained its ability to degrade ulvan. Molecular dynamics simulation analysis and site-directed mutagenesis analysis indicated that the strong rigidity of the disulfide bond between Cys74-Cys102 in the N-terminus is related to its thermostability. In addition, oligosaccharides with disaccharides and tetrasaccharides were the end products of EPL15085. Based on molecular docking and site-directed mutagenesis analysis, Tyr177 and Leu134 are considered to be the crucial residues for enzyme activity. In conclusion, our study identified a new PL28 family of ulvan lyases, EPL15085, with excellent heat resistance that can expand the database of ulvan lyases and provide the possibility to make full use of ulvan.

Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84-CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB. [ABSTRACT FROM AUTHOR]

Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 481 species producing phenylalanine ammonia lyase (PAL) were selected from Uniprot. • PAL6 from Lotus japonicus (LjPAL6) was the most stable PAL in in silico analyses. • Molecular dynamic simulation and docking showed high affinity of LjPAL6 and l -Phe. • LjPAL6 showed K m and K cat values of 0.483 mM, and 7 S−1, respectively. • Overexpression and optimization of Ljpal6 resulted in 83.7 mg/l enzyme production. Abstract The enzyme phenylalanine ammonia lyase (PAL) is of special importance for the treatment of phenylketonuria patients. The aim of this study was to find a stable recombinant PAL with suitable kinetic properties among all natural PAL producing species using in silico and experimental approaches. To find such a stable PAL among 481 natural isoforms, 48,000 of 3-D models were predicted using the Modeller 9.10 program and evaluated by Ramachandran plot. Correlation analysis between Ramachandran plot and the energy of different thermodynamic components indicated that this plot could be an appropriate tool to predict protein stability. Hence, PAL6 from Lotus japonicus (LjPAL6) was selected as a stable isoform. Molecular dynamic (MD) simulation for 50 ns and docking has been conducted for LjPAL6-phenylalanine complex. The best PAL-phenylalanine frame was selected by re-docking with l -phenylalanine (L-Phe) and root-mean-square deviation (RMSD) value. MD simulation showed that the complex has a good stability, depicted by the low RMSD value, binding free energy and hydrogen bindings. Docking results showed that LjPAL6 has a high affinity toward l -Phe according to the low level of binding free energy. By overexpressing Ljpal6 in E. coli BL21, a total of 33.5 mg/l of protein was obtained, which has been increased to 83.7 mg/l via the optimization of LjPAL6 production using response surface methodology. The optimal pH and temperature were 8.5 and 50 °C, respectively. LjPAL6 showed a specific activity of 42 nkat/mg protein, with K m , K cat and K cat / K m values of 0.483 mM, 7 S−1 and 14.5 S−1 mM−1 for l -phe, respectively. In conclusion, finding models with the most reasonable stereo-chemical quality and lowest numbers of steric clashes would result in easier folding. Hence, in silico analyses of bulk data from natural origin will lead one to find an optimal model for in vitro studies and drug design. [ABSTRACT FROM AUTHOR]

Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C–C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR‐based structural elucidation of reaction products reveals 21‐hydroxyprednisone as the major product for prednisone, while the other product is identified as 1‐dehydroadrenosterone obtained due to C–C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C–C bond cleavage products. [ABSTRACT FROM AUTHOR]

Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e.g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunogenicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging. In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage ΦK5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of ΦK5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells. [Display omitted] [ABSTRACT FROM AUTHOR]

Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan. [ABSTRACT FROM AUTHOR]

Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan. [ABSTRACT FROM AUTHOR]

Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5′-deoxyribose phosphate (5′-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5′-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5′-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5′-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs. [ABSTRACT FROM AUTHOR]

Enzymes belonging to the aspartase/fumarase superfamily catalyze elimination of various functional groups from succinate derivatives and play an important role in primary metabolism and aromatic compound degradation. Recently, an aspartase/fumarase superfamily enzyme, CreD, was discovered in cremeomycin biosynthesis. This enzyme catalyzes the elimination of nitrous acid from nitrosuccinate synthesized from aspartate by CreE, a flavin‐dependent monooxygenase. Nitrous acid generated by this pathway is an important precursor of the diazo group of cremeomycin. CreD is the first aspartase/fumarase superfamily enzyme that was reported to catalyze the elimination of nitrous acid, and therefore we aimed to analyze its reaction mechanism. The crystal structure of CreD was determined by the molecular replacement native‐single anomalous diffraction method at 2.18 Å resolution. Subsequently, the CreD‐fumarate complex structure was determined at 2.30 Å resolution by the soaking method. Similar to other aspartase/fumarase superfamily enzymes, the crystal structure of CreD was composed of three domains and formed a tetramer. Two molecules of fumarate were observed in one subunit of the CreD‐fumarate complex. One of them was located in the active site pocket formed by three different subunits. Intriguingly, no histidine residue, which usually functions as a catalytic acid in aspartase/fumarase superfamily enzymes, was found around the fumarate molecule in the active site. Based on the mutational analysis, we propose a catalytic mechanism of CreD, in which Arg325 acts as a catalytic acid. Databases: The crystal structures of CreD and the CreD‐fumarate complex were deposited to PDB under the accession numbers 5XNY and 5XNZ, respectively. Enzymes: Nitrosuccinate lyase CreD, EC4.3 [ABSTRACT FROM AUTHOR]