Summary
The process of obtaining a well‐expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. ...While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His‐tagged Gateway vector, followed by their small‐scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large‐scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.
The process of obtaining a well‐expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semi‐automated pipelines available for cloning, expression, and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening.
Sialic acids comprise a varied group of nine‐carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic ...acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N‐acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5′‐triphosphate to the C6 position of N‐acetylmannosamine to generate N‐acetylmannosamine 6‐phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X‐ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc‐binding site. In spite of the absence of the zinc‐binding site, all of the major structural features of enzymatic activity are conserved.
The enzyme N‐acetylmannosamine kinase (NanK) catalyzes the second step of the bacterial sialic acid catabolic pathway. Here, the structure of F. nucleatum NanK is presented at 2.23 Å resolution.
Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in ...general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium
Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.
The molecular basis underlying T‐cell recognition of MHC molecules presenting altered peptide ligands is still not well–established. A hierarchy of T‐cell activation by MHC class I‐restricted altered ...peptide ligands has been defined using the T‐cell receptor P14 specific for H‐2Db in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H‐2Db in complex with the alanine‐substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H‐2Db/gp33 was strictly enthalpy driven, recognition of the weak agonist H‐2Db/Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H‐2Db/gp33 compared with H‐2Db/Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.
Determining Rieske cluster reduction potentials Brown, Eric N.; Friemann, Rosmarie; Karlsson, Andreas ...
Journal of biological inorganic chemistry,
11/2008, Letnik:
13, Številka:
8
Journal Article
Recenzirano
The Rieske iron–sulfur proteins have reduction potentials ranging from −150 to +400 mV. This enormous range of potentials was first proposed to be due to differing solvent exposure or even protein ...structure. However, the increasing number of available crystal structures for Rieske iron–sulfur proteins has shown this not to be the case. Colbert and colleagues proposed in 2000 that differences in the electrostatic environment, and not structural differences, of a Rieske proteins are responsible for the wide range of reduction potentials observed. Using computational simulation methods and the newly determined structure of
Pseudomonas
sp. NCIB 9816-4 naphthalene dioxygenase Rieske ferredoxin (NDO-F
9816-4
), we have developed a model to predict the reduction potential of Rieske proteins given only their crystal structure. The reduction potential of NDO-F
9816-4
, determined using a highly oriented pyrolytic graphite electrode, was −150 ± 2 mV versus the standard hydrogen electrode. The predicted reduction potentials correlate well with experimentally determined potentials. Given this model, the effect of protein mutations can be evaluated. Our results suggest that the reduction potential of new proteins can be estimated with good confidence from 3D structures of proteins. The structure of NDO-F
9816-4
is the most basic Rieske ferredoxin structure determined to date. Thus, the contributions of additional structural motifs and their effects on reduction potential can be compared with respect to this base structure.
Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three‐component toluene dioxygenase enzyme ...system consisting of a reductase (ReductaseTOL), a ferredoxin (FerredoxinTOL) and a Rieske non‐heme iron dioxygenase (OxygenaseTOL). The three components and the apoenzyme of the dioxygenase (apo‐OxygenaseTOL) were overexpressed, purified and crystallized. ReductaseTOL diffracts to 1.8 Å and belongs to space group P41212, with unit‐cell parameters a = b = 77.1, c = 156.3 Å. FerredoxinTOL diffracts to 1.2 Å and belongs to space group P21, with unit‐cell parameters a = 30.5, b = 52.0, c = 30.95 Å, β = 113.7°. Apo‐OxygenaseTOL and OxygenaseTOL diffract to 3.2 Å and belong to space group P4332, with unit‐cell parameters a = 235.9 Å and a = 234.5 Å, respectively.
The three-dimensional structure of thioredoxin from
Trypanosoma brucei brucei has been determined at 1.4 Å resolution. The overall structure is more similar to that of human thioredoxin than to any ...other thioredoxin structure. The most striking difference to other thioredoxins is the absence of a buried carboxylate behind the active site cysteines. Instead of the common Asp, there is a Trp that binds an ordered water molecule probably involved in the protonation/deprotonation of the more buried cysteine during catalysis. The conserved Trp in the WCGPC sequence motif has an exposed position that can interact with target proteins.
Acidovorax (formerly Pseudomonas) sp. strain JS42 utilizes 2-nitrotoluene as sole carbon, nitrogen, and energy source. 2-Nitrotoluene 2,3-dioxygenase (2NTDO) catalyzes the initial step in ...2-nitrotoluene degradation by converting 2-nitrotoluene to 3-methylcatechol. In this study, we identified specific amino acids at the active site that control specificity. The residue at position 350 was found to be critical in determining both the enantiospecificity of 2NTDO with naphthalene and the ability to oxidize the ring of mononitrotoluenes. Substitution of Ile350 by phenylalanine resulted in an enzyme that produced 97% (+)-(1R, 2S)-cis-naphthalene dihydrodiol, in contrast to the wild type, which produced 72% (+)-(1R, 2S)-cis-naphthalene dihydrodiol. This substitution also severely reduced the ability of the enzyme to produce methylcatechols from nitrotoluenes. Instead, the methyl group of each nitrotoluene isomer was preferentially oxidized to form the corresponding nitrobenzyl alcohol. Substitution of a valine at position 258 significantly changed the enantiospecificity of 2NTDO (54% (-)-(1S, 2R)-cis-naphthalene dihydrodiol formed from naphthalene) and the ability of the enzyme to oxidize the aromatic ring of nitrotoluenes. Based on active site modeling using the crystal structure of nitrobenzene 1,2 dioxygenase from Comamonas sp. JS765, Asn258 appears to contribute to substrate specificity through hydrogen bonding to the nitro group of nitrotoluenes.
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