Publications
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Tuveng, Tina Rise; Rothweiler, Ulli; Udatha, D.B.R.K. Gupta; Vaaje-Kolstad, Gustav; Smalås, Arne O. & Eijsink, Vincent
(2017).
Structure and function of a CE4 deacetylase isolated from a marine environment.
PLOS ONE.
12(11).
doi:
10.1371/journal.pone.0187544.
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Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.
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Leo, Jack Christopher; Oberhettinger, Philipp; Yoshimoto, Shogo; Udatha, D.B.R.K. Gupta; Morth, Jens Preben & Schutz, Monika
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(2016).
Secretion of the Intimin passenger domain is driven by protein folding.
Journal of Biological Chemistry.
ISSN 0021-9258.
291(38),
p. 20096–20112.
doi:
10.1074/jbc.M116.731497.
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Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli. It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane β-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the β-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction. © 2016 American Society for Biochemistry and Molecular Biology
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Fan, Enguo; Chauhan, Nandini; Udatha, D.B.R.K. Gupta; Leo, Jack Christopher & Linke, Dirk
(2016).
Type V secretion systems in bacteria.
In Kudva, Indira T.; Cornick, Nancy A.; Plummer, Paul J.; Zhang, Qijing; Nicholson, Tracy L.; Bannantine, John P. & Bellaire, Bryan H. (Ed.),
Virulence Mechanisms of Bacterial Pathogens.
ASM International.
ISSN 9781555819279.
p. 305–335.
doi:
10.1128/microbiolspec.VMBF-0009-2015.
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Udatha, Dasaradhi B R K G; Evangelos, Topakas; Salazar, Margarita; Olsson, Lisbeth; Andersen, Mikael R. & Panagiotou, Gianni
(2015).
Deciphering the signaling mechanisms of the plant cell wall degradation machinery in Aspergillus oryzae.
BMC Systems Biology.
9:77,
p. 1–20.
doi:
10.1186/s12918-015-0224-5.
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Background The gene expression and secretion of fungal lignocellulolytic enzymes are tightly controlled at the transcription level using independent mechanisms to respond to distinct inducers from plant biomass. An advanced systems-level understanding of transcriptional regulatory networks is required to rationally engineer filamentous fungi for more efficient bioconversion of different types of biomass. Results In this study we focused on ten chemically defined inducers to drive expression of cellulases, hemicellulases and accessory enzymes in the model filamentous fungus Aspergillus oryzae and shed light on the complex network of transcriptional activators required. The chemical diversity analysis of the inducers, based on 186 chemical descriptors calculated from the structure, resulted into three clusters, however, the global, metabolic and extracellular protein transcription of the A. oryzae genome were only partially explained by the chemical similarity of the enzyme inducers. Genes encoding enzymes that have attracted considerable interest such as cellobiose dehydrogenases and copper-dependent polysaccharide mono-oxygenases presented a substrate-specific induction. Several homology-model structures were derived using ab-initio multiple threading alignment in our effort to elucidate the interplay of transcription factors involved in regulating plant-deconstructing enzymes and metabolites. Systematic investigation of metabolite-protein interactions, using the 814 unique reactants involved in 2360 reactions in the genome scale metabolic network of A. oryzae, was performed through a two-step molecular docking against the binding pockets of the transcription factors AoXlnR and AoAmyR. A total of six metabolites viz., sulfite (H 2 SO 3 ), sulfate (SLF), uroporphyrinogen III (UPGIII), ethanolamine phosphate (PETHM), D-glyceraldehyde 3-phosphate (T3P1) and taurine (TAUR) were found as strong binders, whereas the genes involved in the metabolic reactions that these metabolites appear were found to be significantly differentially expressed when comparing the inducers with glucose. Conclusions Based on our observations, we believe that specific binding of sulfite to the regulator of the cellulase gene expression, AoXlnR, may be the molecular basis for the connection of sulfur metabolism and cellulase gene expression in filamentous fungi. Further characterization and manipulation of the regulatory network components identified in this study, will enable rational engineering of industrial strains for improved production of the sophisticated set of enzymes necessary to break-down chemically divergent plant biomass.
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Udatha, Dasaradhi B R K G; Madsen, Karina Marie; Panagiotou, Gianni & Olsson, Lisbeth
(2015).
Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum.
Journal of Structural Biology.
ISSN 1047-8477.
190(3),
p. 314–327.
doi:
10.1016/j.jsb.2015.04.009.
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The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silico analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases.
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Published
Apr. 27, 2015 8:24 AM
- Last modified
Mar. 21, 2017 11:09 AM