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Stochastic processes are widely used to analyze degradation data, and the Gaussian process is a particularly common one. In this article, we propose a robust statistical model using a Student-t process to assess the lifetime information of highly reliable products. This model is statistically plausible and demonstrates a substantially improved fit when applied to real data. A computationally accurate approach is proposed to calculate the first-passage-time density function of the Student-t degradation-based process; related properties are investigated as well. In addition, this article provides parameter estimation using the EM-type algorithm and a simple model-checking procedure to evaluate the appropriateness of the model assumptions. Several case studies are performed to demonstrate the flexibility and applicability of the proposed model with random effects and explanatory variables. Technical details, datasets, and R codes are available as . [ABSTRACT FROM AUTHOR]

XynCDBFV from Neocallimastix patriciarum is among the most effective xylanases and holds great potentials in a wide variety of industrial applications. In the present study, several active site residues were modified referring to the instrumental information of the complex structure of XynCDBFV and xylooligosaccharides. Among the 12 single active site mutants, W125F and F163W show increased activity comparing to the wild-type protein. The double mutant W125F/F163W was then generated which displayed nearly 20 % increase in the enzyme activity. Although W125F/F163W showed 5 °C reduction in the optimal temperature, it still preserves similar thermostability and is more active than the wild-type enzyme at temperatures lower than 60 °C. These properties make the double mutant a suitable candidate for commercial applications that involve lower operating temperatures. Furthermore, we investigated the effect of N-glycosylation on the thermostability of XynCDBFV when expressed in the yeast strain Pichia pastoris for industrial use. Two potential glycosylation sites (Asn-37 and Asn-88) were examined, and their roles in enzyme performance were validated. We found that the N-glycosylations of XynCDBFV are related to both catalytic activity and heat stability, with Asn-37 motif playing a dominant role. Collectively, the enzymatic properties of XynCDBFV were improved by molecular engineering, and the influences of N-glycosylations on the enzyme have been clearly elucidated herein. [ABSTRACT FROM AUTHOR]

Abstract: The thermostable 1,3–1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80Å to 2.25Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3–1,4-β-glucotriose complex, the sugar bound to the −1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3–1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering. [Copyright &y& Elsevier]

Cellulase 12A from Thermotoga maritima ( TmCel12A) is a hyperthermostable β-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k and K values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application. [ABSTRACT FROM AUTHOR]

1,3-1,4-β- d-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β- d-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity. [ABSTRACT FROM AUTHOR]

Highlights: [•] E. coli phytase gained higher activity via adopting active site residues from Citrobacter phytases. [•] An N-glycosylation motif at substrate entry site of EcAppA was disrupted to enhance catalytic activity. [•] N-glycosylation motifs from thermophilic CbAppA confer EcAppA higher thermostability. [ABSTRACT FROM AUTHOR]

Abstract: We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics. [ABSTRACT FROM AUTHOR]

Abstract: We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics. [ABSTRACT FROM AUTHOR]

Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β- d -glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris . Here, the crystal structure and complex structures of P. pastoris -expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product. [ABSTRACT FROM AUTHOR]

We report the first X-ray structure of the unique 'head-to-middle' monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase. [ABSTRACT FROM AUTHOR]

We report the first X-ray structure of the unique 'head-to-middle' monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase. [ABSTRACT FROM AUTHOR]

A thermophilic glycoside hydrolase family 16 (GH16) β-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 Å resolution. The enzyme folds into a classic GH16 β-jellyroll architecture which consists of two β-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner β-sheet. Two Bis–Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 β-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris , encouraging its further commercial applications. [ABSTRACT FROM AUTHOR]

β-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (β/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications. [ABSTRACT FROM AUTHOR]

Abstract: The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29Å to 2.40Å resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers β-glucosyl and β-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues. [Copyright &y& Elsevier]

The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (β/α)8-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme-substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the −3, −2 and −1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the −3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (β/α)8-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem. [ABSTRACT FROM AUTHOR]

Abstract: Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general. [Copyright &y& Elsevier]

Die irreguläre Terpensynthase Lavandulyldiphosphatsynthase (LPPS) produziert die Vorstufe des kommerziell wichtigen Lavendelduftstoffs Lavandulol durch einen ungewöhnlichen “Kopf ‐ zur ‐ Mitte” ‐ Mechanismus. E. Oldfield, R. ‐ T. Guo, Y. Ma et al. präsentieren in der Zuschrift auf S. 4799 ff. die erste Struktur von LPPS im Komplex mit seinem Substrat. Die schmetterlingsförmige Struktur ähnelt der von Proteinen, die an der Biosynthese bakterieller Zellwände beteiligt sind, und führt zu einem einzigartigen Kondensationsmechanismus, der von allgemeinem Interesse im Kontext der Terpensynthese ist. [ABSTRACT FROM AUTHOR]

The article presents a diagram related to the article "Structure and Function of a "Head-to-Middle" Prenyltransferase: Lavandulyl Diphosphate Synthase" by Y. Ma and colleagues in the current issue.