Scenario-based approach to analysis of travel time reliability with traffic simulation models

This study established a conceptual framework for capturing the probabilistic nature of travel times with the use of existing traffic simulation models. The framework features three components: scenario manager, traffic simulation models, and trajectory processor. The scenario manager captures exogenous sources of variation in travel times through external scenarios consistent with real-world roadway disruptions. The traffic simulation models then produce individual vehicle trajectories for input scenarios while further introducing randomness that stems from endogenous sources of variation. Finally, the trajectory processor constructs distributions of travel time either for each scenario or for multiple scenarios to allow users to investigate scenario-specific impact on variability in travel times and overall system reliability. Within this framework, the paper discusses methodologies for performing scenario-based reliability analysis that focuses on (a) approaches to obtaining distributions of travel times from scenario-specific outputs and (b) issues and practices associated with designing and generating input scenarios. The proposed scenario-based approach was applied to a real-world network to show detailed procedures, analysis results, and their implications

Structural basis of high-order oligomerization of the cullin-3 adaptor SPOP.

Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5 Å resolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination

In vivo and in vitro FMN prenylation and (de)carboxylase activation under aerobic conditions

Prenylated FMN (prFMN) is a newly discovered redox cofactor required for activity of the large family of reversible UbiD (de)carboxylases involved in biotransformation of aromatic, heteroaromatic, and unsaturated aliphatic acids (White et al., 2015). Despite the growing demand for decarboxylases in the pulp/paper industry and in forest biorefineries, the vast majority of UbiD-like decarboxylases remain uncharacterized. Functional characterization of the novel UbiD decarboxylases is hindered by the lack of prFMN generating system. prFMN cofactor is synthesized by the UbiX family of FMN prenyltransferases, which use reduced FMN as substrate under anaerobic conditions and dimethylallyl-monophosphate (DMAP) as the prenyl group donor. Here, we report the in vivo and in vitro biosynthesis of prFMN and UbiD activation under aerobic conditions. For in vivo biosynthesis, we used newly discovered UbiX proteins from Salmonella typhimurium and Klebsiella pneumonia, which activated ferulic acid UbiD decarboxylase Fdc1 from Aspergillus niger under aerobic conditions (0.5-1.5 U/mg). For in vitro biosynthesis of prFMN and UbiD activation, we established a one-pot enzyme cascade system that uses prenol, polyphosphate, formate, and riboflavin as starting substrates and (re)generates DMAP, ATP, FMN and NADH. The system contains 6 different enzymes: prenol kinase, polyphosphate kinase, formate dehydrogenase, FMN reductase, riboflavin kinase and FMN prenyltransferase. Under aerobic conditions, this system showed up to 80% conversion of FMN to prFMN and generated active Fdc1 decarboxylase (0.2-1 U/mg). Thus, both systems represent robust approaches for in vivo and in vitro prFMN biosynthesis and UbiD activation under aerobic conditions. The developed FMN prenylation systems will facilitate the exploration and biochemical characterization of UbiD-like decarboxylases and their applications in biocatalysis. Please click Additional Files below to see the full abstract

Refining and mining the phylogeny of Glycoside Hydrolase Family 74 via structure-function analysis

Sustained interest in the use of carbohydrates from plant cell walls, coupled with the advancement of high-throughput (meta)genomic sequencing, has led to the discovery of an overwhelming number of predicted carbohydrate-active enzymes (CAZymes) in the last decade. The CAZy database provides a powerful framework for the study of CAZymes, including Glycoside Hydrolases (GHs), by enabling the prediction of key enzyme features such as 3-D fold, catalytic residues, catalytic mechanism, and – with certain limitations – substrate specificity. Refined phylogenetic analyses contribute to increasing the accuracy of predictions by further clustering proteins into sub-families (1, 2). However, reliable prediction of substrate specificity for newly discovered GHs remains a challenge due to a general lack of in-depth biochemical and structural characterization across the existing phylogenetic diversity. Glycoside Hydrolase family 74 (GH74) comprises endo-glucanases, many of which have predominant activity toward xyloglucan, a highly branched plant cell wall matrix glycan. To better delineate overall substrate specificity, backbone cleavage position, and endo-dissociative vs. endo-processive hydrolytic modes, a broad-based structure-function analysis of GH74 guided by molecular phylogeny was performed. Seven sub-families were discerned, which grouped nearly 40% of the current \u3e300 GH74 sequences in the public CAZy database. Thirty one GH74 members were targeted for further investigation based on their phylogenetic position and unique primary structural features identified during manual curation. The biochemical characterization of 18 recombinant GH74s revealed key sequence features governing xyloglucan backbone cleavage sites and highlighted clear phylogenetic differences between endo-dissociative and endo-processive enzymes. Commensurate with previous studies (3), site-directed mutagenesis of key active-site tryptophan residues defined their essential contributions to processivity on the soluble polysaccharide substrate. Six new GH74 tertiary structures (apo and/or in complex with xylogluco-oligosaccharides) were determined that further resolved the contribution of active-site loops in modulating the size of oligosaccharide products released by individual subfamily members. Refining the correlation between phylogeny and enzyme structure-function properties in GH74 significantly enhances the prediction of catalytic ability, highlights key steps in the evolution of function in the family, and ultimately informs applications in biomass conversion. 1. Stam MR, Danchin EGJ, Rancurel C, Coutinho PM, Henrissat B. 2006. Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase-related proteins. Protein Engineering Design & Selection 19:555-562. 2. Aspeborg H, Coutinho PM, Wang Y, Brumer H, Henrissat B. 2012. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). Bmc Evolutionary Biology 12. 3. Matsuzawa T, Saito Y, Yaoi K. 2014. Key amino acid residues for the endo-processive activity of GH74 xyloglucanase. FEBS Lett 588:1731-8

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Sequence and structural analysis of BTB domain proteins

BACKGROUND: The BTB domain (also known as the POZ domain) is a versatile protein-protein interaction motif that participates in a wide range of cellular functions, including transcriptional regulation, cytoskeleton dynamics, ion channel assembly and gating, and targeting proteins for ubiquitination. Several BTB domain structures have been experimentally determined, revealing a highly conserved core structure. RESULTS: We surveyed the protein architecture, genomic distribution and sequence conservation of BTB domain proteins in 17 fully sequenced eukaryotes. The BTB domain is typically found as a single copy in proteins that contain only one or two other types of domain, and this defines the BTB-zinc finger (BTB-ZF), BTB-BACK-kelch (BBK), voltage-gated potassium channel T1 (T1-Kv), MATH-BTB, BTB-NPH3 and BTB-BACK-PHR (BBP) families of proteins, among others. In contrast, the Skp1 and ElonginC proteins consist almost exclusively of the core BTB fold. There are numerous lineage-specific expansions of BTB proteins, as seen by the relatively large number of BTB-ZF and BBK proteins in vertebrates, MATH-BTB proteins in Caenorhabditis elegans, and BTB-NPH3 proteins in Arabidopsis thaliana. Using the structural homology between Skp1 and the PLZF BTB homodimer, we present a model of a BTB-Cul3 SCF-like E3 ubiquitin ligase complex that shows that the BTB dimer or the T1 tetramer is compatible in this complex. CONCLUSION: Despite widely divergent sequences, the BTB fold is structurally well conserved. The fold has adapted to several different modes of self-association and interactions with non-BTB proteins

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

| openaire: EC/H2020/648925/EU//BHIVEAcetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono-or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.Peer reviewe