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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024. Published by Elsevier Ltd.)

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.

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., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Chondroitinase ABC-type I (CSase ABC I), which can digest both chondroitin sulfate (CS) and dermatan sulfate (DS) in an endolytic manner, is an essential tool in structural and functional studies of CS/DS. Although a few CSase ABC I have been identified from bacteria, the substrate-degrading pattern and regulatory mechanisms of them have rarely been investigated. Herein, two CSase ABC I, IM3796 and IM1634, were identified from the intestinal metagenome of CS-fed mice. They show high sequence homology (query coverage: 88.00%, percent identity: 90.10%) except for an extra peptide (Met 1 -His 109 ) at the N-terminus in IM1634, but their enzymatic properties are very different. IM3796 prefers to degrade 6-O-sulfated GalNAc residue-enriched CS into tetra- and disaccharides. In contrast, IM1634 exhibits nearly a thousand times more activity than IM3796 and can completely digest CS/DS with various sulfation patterns to produce disaccharides, unlike most CSase ABC I. Structure modeling showed that IM3796 did not contain an N-terminal domain composed of two β-sheets, which is found in IM1634 and other CSase ABC I. Furthermore, deletion of the N-terminal domain (Met 1 -His 109 ) from IM1634 caused the enzymatic properties of the variant IM1634-T109 to be similar to those of IM3796, and conversely, grafting this domain to IM3796 increased the similarity of the variant IM3796-A109 to IM1634. In conclusion, the comparative study of the new CSase ABC I provides two unique tools for CS/DS-related studies and applications and, more importantly, reveals the critical role of the N-terminal domain in regulating the substrate binding and degradation of these enzymes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

We report herein, the discovery of BMS-737 (compound 33) as a potent, non-steroidal, reversible small molecule inhibitor demonstrating 11-fold selectivity for CYP17 lyase over CYP17 hydroxylase, as well as a clean xenobiotic CYP profile for the treatment of castration-resistant prostate cancer (CRPC). Extensive SAR studies on the initial lead 1 at three different regions of the molecule resulted in the identification of BMS-737, which demonstrated a robust 83% lowering of testosterone without any significant perturbation of the mineralocorticoid and glucocorticoid levels in cynomologous monkeys in a 1-day PK/PD study., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

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 3'-sialyllactose (3SL) synthesis from N-acetyl- d-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5'-triphosphate, via the reactions of cytidine 5'-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., (© 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)

Desulfonation of isethionate by the bacterial glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslA) generates sulfite, a substrate for respiration that in turn produces the disease-associated metabolite hydrogen sulfide. Here, we present a 2.7 Å resolution X-ray structure of wild-type IslA from Bilophila wadsworthia with isethionate bound. In comparison with other GREs, alternate positioning of the active site β strands allows for distinct residue positions to contribute to substrate binding. These structural differences, combined with sequence variations, create a highly tailored active site for the binding of the negatively charged isethionate substrate. Through the kinetic analysis of 14 IslA variants and computational analyses, we probe the mechanism by which radical chemistry is used for C-S bond cleavage. This work further elucidates the structural basis of chemistry within the GRE superfamily and will inform structure-based inhibitor design of IsIA and thus of microbial hydrogen sulfide production., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: XG, AS, SK, GI, OW, UE, ST, AG, JJ, JÅ, DA, MS No competing interests declared, (© 2021, Guo et al.)

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.

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., Competing Interests: The authors declare no conflict of interest.

During alcoholic fermentation of grape sugars, wine yeasts produce a range of secondary metabolites that play an important role in the aroma profile of wines. In this study, we have explored the ability of a large number of wine yeast strains to modulate wine aroma composition, focusing on the release of the "fruity" thiols 3-mercaptohexan-1-ol (3-MH) and 4-mercapto-4-methylpentan-2-one (4-MMP) from their respective cysteinylated nonvolatile precursors. The role of the yeast gene IRC7 in thiol release has been well established, and it has been shown that a 38-bp deletion found in many wine strains cause them to express a truncated version of Irc7p that does not possess cysteine- S -conjugate β-lyase activity. In our data, we find that IRC7 allele length alone does not fully explain the capacity of a strain to release thiols. Screening of a large number of strains coupled with analysis of genomic sequence data allowed us to identify several previously undescribed single-nucleotide polymorphisms (SNPs) in IRC7 that, when coupled with allele length, more robustly explain the ability of a particular yeast strain to release thiols from their cysteinylated precursors. We also demonstrate that allelic variation of IRC7 not only affects the release of thiols but modulates the formation of negative volatile sulfur compounds from the amino acid cysteine. The results of this study provide winemakers with an improved understanding of the genetic determinants that affect wine aroma and flavor, which can be used to guide the choice of yeast strains that are fit for purpose. IMPORTANCE Volatile sulfur compounds contribute to wine aromas that may be considered pleasant, such as "tropical," "passionfruit," and "guava," as well as aromas that are considered undesirable, such as "rotten eggs," "onions," and "sewer." During fermentation, wine yeasts release some of these compounds from odorless precursor molecules, a process that is most efficient when performed by yeasts that express active forms of the protein Irc7p. We show that most wine yeasts carry mutations that reduce activity of this protein, affecting the formation of volatile sulfur compounds that impart both pleasant and unpleasant aromas. The results provide winemakers with guidance on the choice of yeasts that can emphasize or deemphasize this particular contribution to wine quality., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

During the course of an investigation of targeted inhibition of DNA polymerase beta (pol β) lyase activity using small molecules, we observed the formation of an aldimine between (2-formyl)phenylphosphonic acid (2FPP) and butylamine under basic aqueous conditions; complete deprotonation of the phosphonate group was required to stabilize the imine product. Results of computational docking studies suggested that the reaction of Lys-72 on the lyase active site with an aldehyde group could be facilitated by a proximal phosphonate, not only because of the phosphonate's ability to mimic phosphate interacting with the DNA binding site, but also because of its ability to shield the imine against hydrolysis. Novel pol β lyase inhibitors were thus prepared using a 2FPP analogue with an amine linker; P-C bond formation in synthesis of this intermediate was possible with an unprotected aldehyde using palladium-catalyzed, microwave-assisted Michaelis-Arbuzov chemistry. These compounds, and structurally related derivatives lacking the aldehyde or phosphonate, were evaluated in an assay for pol β, to assess their potential for inhibition of pol β.

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., (© 2018 Federation of European Biochemical Societies.)

DNA polymerase β plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA polymerase β contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA polymerases., (© 2019 Elsevier Inc. All rights reserved.)

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 3901 T 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 Ca 2+ 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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (© 2018 Federation of European Biochemical Societies.)

GlcUAβ1-3GalNAc(4S,6S) (E unit)-rich domains have been shown to play key roles in various biological functions of chondroitin sulfate (CS). However, an enzyme that can specifically isolate such domains through the selective digestion of other domains in polysaccharides has not yet been reported. Here, we identified a glycosaminoglycan lyase from a marine bacterium Vibrio sp. FC509. This enzyme efficiently degraded hyaluronic acid (HA) and CS variants, but not E unit-rich CS-E, into unsaturated disaccharides; therefore, we designated this enzyme a CS-E-resisted HA/CS lyase (HCLase Er). We isolated a series of resistant oligosaccharides from the final product of a low-sulfated CS-E exhaustively digested by HCLase Er and found that the E units were dramatically accumulate in these resistant oligosaccharides. By determining the structures of several resistant tetrasaccharides, we observed that all of them possessed a Δ 4,5 HexUAα1-3GalNAc(4S,6S) at their non-reducing ends, indicating that the disulfation of GalNAc abrogates HCLase Er activity on the β1-4 linkage between the E unit and the following disaccharide. Δ 4,5 HexUAα1-3GalNAc(4S,6S)β1-4GlcUAβ1-3GalNAc(4S,6S) was most strongly resistant to HCLase Er. To our knowledge, this study is the first reporting a glycosaminoglycan lyase specifically inhibited by both 4- O - and 6- O -sulfation of GalNAc. Site-directed and truncation mutagenesis experiments indicated that HCLase Er may use a general acid-base catalysis mechanism and that an extra domain (Gly 739 -Gln 796 ) is critical for its activity. This enzyme will be a useful tool for structural analyses and for preparing bioactive oligosaccharides of HA and CS variants, particularly from E unit-rich CS chains., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Given prostate cancer is driven, in part, by its responsiveness to androgens, treatments historically employ methods for their removal from circulation. Approaches as crude as castration, and more recently blockade of androgen synthesis or receptor binding, are still of limited use long term, since other steroids of adrenal origin or tumor origin can supersede that role as the 'castration resistant' tumor re-emerges. Broader inhibition of steroidogenesis using relatively nonselective P450 inhibitors such as ketoconazole is not an alternative since a general disruption of steroid biosynthesis is neither safe nor effective. The recent emergence of drugs more selectively targeting CYP17 have been more effective, and yet extension of life has been on the scale of months rather than years. It is now becoming clear this shortcoming arises from the adaptive capabilities of many tumors to initiate local steroid synthesis and/or become responsive to novel early pathway adrenal steroids that are synthesized when lyase activity is not selectively blocked, and ACTH rises in the face of declining cortisol feedback. Abiraterone has been described as a lyase selective inhibitor, yet its use still requires co-administration of prednisone to suppress such a rise of ACTH and fall in cortisol. So is creation of a selective lyase inhibitor even possible? Can C19 steroid production be achieved without a prominent decline in cortisol and corresponding rise in ACTH? Decades of scientific study of CYP17 in humans and nonhuman primates, as well as nature's own experiments of gene mutations in humans, reveal 'true' or 'isolated' 17,20 lyase deficiency does quite selectively prevent C19 steroid biosynthesis whereas simple 17 hydroxylase deficiency also suppresses cortisol. We propose these known outcomes of natural mutations should be used to guide analysis of clinical trials and long term outcomes of CYP17 targeted drugs. In this review, we use that framework to re-evaluate the basic and clinical outcomes of many compounds being used or in development for treatment of castration resistant prostate cancer. Specifically, we include the nonselective drug ketoconazole, and then the CYP17 targeted drugs abiraterone, orteronel (TAK-700), galaterone (TOK-001), and seviteronel (VT-464). Using this framework, we can fully discriminate the clinical outcomes for ketoconazole, a drug with broad specificity, yet clinically ineffective, from that of abiraterone, the first CYP17 targeted therapy that is limited by its need for prednisone co-therapy. We also can identify potential next generation CYP17 targeted drugs now emerging that show signs of being far more 17,20 lyase selective. We conclude that a future for improved therapy without substantial cortisol decline, thus avoiding prednisone co-administration, seems possible at long last., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A series of potential active-site sphingosine-1-phosphate lyase (S1PL) inhibitors have been designed from scaffolds 1 and 2, arising from virtual screening using the X-ray structures of the bacterial (StS1PL) and the human (hS1PL) enzymes. Both enzymes are very similar at the active site, as confirmed by the similar experimental kinetic constants shown by the fluorogenic substrate RBM13 in both cases. However, the docking scoring functions used probably overestimated the weight of electrostatic interactions between the ligands and key active-site residues in the protein environment, which may account for the modest activity found for the designed inhibitors. In addition, the possibility that the inhibitors do not reach the enzyme active site should not be overlooked. Finally, since both enzymes show remarkable structural differences at the access channel and in the proximity to the active site cavity, caution should be taken when designing inhibitors acting around that area, as evidenced by the much lower activity found in StS1PL for the potent hS1PL inhibitor D., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions.

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Efforts to identify a potent, reversible, nonsteroidal CYP17A1 lyase inhibitor with good selectivity over CYP17A1 hydroxylase and CYPs 11B1 and 21A2 for the treatment of castration-resistant prostate cancer (CRPC) culminated in the discovery of BMS-351 (compound 18), a pyridyl biaryl benzimidazole with an excellent in vivo profile. Biological evaluation of BMS-351 at a dose of 1.5 mg in castrated cynomolgus monkeys revealed a remarkable reduction in testosterone levels with minimal glucocorticoid and mineralcorticoid perturbation. Based on a favorable profile, BMS-351 was selected as a candidate for further preclinical evaluation.

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity., (© 2015 Wiley Periodicals, Inc.)

Enzymes within a family often catalyze different reactions. In some cases, this variety stems from different catalytic machinery, but in other cases the machinery is identical; nevertheless, the enzymes catalyze different reactions. In this review, we examine the subset of α/β-hydrolase fold enzymes that contain the serine-histidine-aspartate catalytic triad. In spite of having the same protein fold and the same core catalytic machinery, these enzymes catalyze seventeen different reaction mechanisms. The most common reactions are hydrolysis of C-O, C-N and C-C bonds (Enzyme Classification (EC) group 3), but other enzymes are oxidoreductases (EC group 1), acyl transferases (EC group 2), lyases (EC group 4) or isomerases (EC group 5). Hydrolysis reactions often follow the canonical esterase mechanism, but eight variations occur where either the formation or cleavage of the acyl enzyme intermediate differs. The remaining eight mechanisms are lyase-type elimination reactions, which do not have an acyl enzyme intermediate and, in four cases, do not even require the catalytic serine. This diversity of mechanisms from the same catalytic triad stems from the ability of the enzymes to bind different substrates, from the requirements for different chemical steps imposed by these new substrates and, only in about half of the cases, from additional hydrogen bond partners or additional general acids/bases in the active site. This detailed analysis shows that binding differences and non-catalytic residues create new mechanisms and are essential for understanding and designing efficient enzymes.

Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pK(a) calculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid. However, the reaction mechanism can also be explained by an antiperiplanar trans-elimination reaction, in which Lys108 abstracts a proton from the C5 atom without the help of simultaneous proton donation by an acidic residue. An acidified water molecule completes the anti β-elimination reaction by protonating the O4 atom of the substrate. Both the C5 hydrogen and C4 hydroxyl groups of the substrate must be orientated in axial configurations, as for galacturonic acid, for this to be possible. The wild-type C. bescii PL3 displays a pH optimum that is lower than that of Bacillus subtilis PL1 according to activity measurements, indicating that C. bescii PL3 has acquired a lower pH optimum by utilizing lysine instead of arginine as the catalytic base, as well as by lowering the pK(a) of the catalytic base in a unique active-site environment.

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pigmentation of light-harvesting phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apophycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965-983)., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Lyase-catalyzed degradation has been proposed as a more cell-friendly alternative to dissolution of alginate gels than using chelating agents. In this study, we investigated the effect of lyase specificity on degradation of alginate gels, including the effect of crosslinking ions with different affinity for the polymer. Degradation kinetics and products were analyzed. In particular, the degradation products were characterized using novel methods for alginate sequence determination by chromatography. Lyase-catalyzed gel disruption worked well for gels crosslinked with calcium, but was less effective when barium was included in the gel formulation. The importance of crosslinking of long G-blocks in maintaining the structural integrity of the gels was identified. The failure to degrade these long G-blocks, either due to protection of the G-blocks by strong ionic crosslinking or due to lack of lyase activity on G-G linkages, resulted in retained resistance to mechanical disruption of the gel., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)