Identification of the residues that are responsible for improving the activities of cyanobacterial enzymes for hydrocarbon biosynthesis

Cyanobacteria can produce hydrocarbons corresponding to diesel fuels via a reaction catalyzed by two enzymes, acyl-ACP reductase (AAR) and aldehyde deformylating oxygenase (ADO). Because Escherichia coli coexpressing these enzymes can produce and secrete hydrocarbons, both AAR and ADO are key enzymes for hydrocarbon biosynthesis. However, the activities of AAR and ADO are low. Therefore, construction of highly active mutants of AAR and ADO is necessary for industrial application of these enzymes for producing hydrocarbons. Our purpose in this study is to identify the residues that are responsible for improving the activities of AAR and ADO. First, we compared the activity of AARs from several cyanobacteria and detected a highly active AAR. Second, we introduced various single amino acid substitutions into a poorly active AAR, to make its sequence close to that of the highly active AAR. When we constructed and analyzed 40 mutants of AAR, we succeeded in identifying the residues that are important for high activity of AAR and those important for high expression level of soluble AAR (Figure. 1). Combination of single mutations greatly improved the aldehyde productivity. Similarly, we also identified the residues that are important for high activity of ADO and those important for high expression level of soluble ADO (Figure. 2). Mutational analysis of ADO revealed that high productivity of hydrocarbons can be achieved by increasing both the activity and amount of soluble ADO. Our data will be useful for producing higher amount of hydrocarbons using highly active mutants of AAR and ADO created by protein engineering. Please click Additional Files below to see the full abstract

Leu628 of the KIX domain of CBP is a key residue for the interaction with the MLL transactivation domain

AbstractPhysical interaction between the transactivation domain (TAD) of the mixed-lineage leukemia protein (MLL) and the KIX domain of the cyclic-AMP response element binding protein (CREB) binding protein (CBP) is necessary for MLL-mediated transcriptional activation. We show by alanine-scanning mutagenesis that hydrophobic surface residues of KIX, especially L628, are energetically important for binding the MLL TAD. NMR studies of the KIX-L628A mutant suggest that L628 plays a crucial role in conformational transitions at the MLL binding site, necessary for high affinity interactions with MLL. Unexpectedly, MLL also binds to the c-Myb/phosphorylated kinase-inducible domain of CREB (pKID) site of KIX, highlighting the complex nature of interactions involving intrinsically disordered transcriptional activators.Structured summaryMINT-8044564, MINT-8044580, MINT-8044598, MINT-8044616, MINT-8044634, MINT-8044656:Cbp (uniprotkb:P45481) and MLL (uniprotkb:Q03164) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-8044696:Cbp (uniprotkb:P45481) and MLL (uniprotkb:Q03164) bind (MI:0407) by nuclear magnetic resonance (MI:0077

Comparison of aldehyde-producing activities of cyanobacterial acyl-(acyl carrier protein) reductases

Additional file 1: Table S1. Cyanobacterial AARs found by the BLAST search. The group number in the phylogenetic tree is shown in the first column. In each group, the cyanobacterial strains are listed in the same order as in Fig. 1. Twelve representative AARs used in the present study are shown in bold

Oligomeric Hsp33 with enhanced chaperone activity

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress

The Wako-Saitô-Muñoz-Eaton Model for Predicting Protein Folding and Dynamics

Despite the recent advances in the prediction of protein structures by deep neutral networks, the elucidation of protein-folding mechanisms remains challenging. A promising theory for describing protein folding is a coarse-grained statistical mechanical model called the Wako-Saitô-Muñoz-Eaton (WSME) model. The model can calculate the free-energy landscapes of proteins based on a three-dimensional structure with low computational complexity, thereby providing a comprehensive understanding of the folding pathways and the structure and stability of the intermediates and transition states involved in the folding reaction. In this review, we summarize previous and recent studies on protein folding and dynamics performed using the WSME model and discuss future challenges and prospects. The WSME model successfully predicted the folding mechanisms of small single-domain proteins and the effects of amino-acid substitutions on protein stability and folding in a manner that was consistent with experimental results. Furthermore, extended versions of the WSME model were applied to predict the folding mechanisms of multi-domain proteins and the conformational changes associated with protein function. Thus, the WSME model may contribute significantly to solving the protein-folding problem and is expected to be useful for predicting protein folding, stability, and dynamics in basic research and in industrial and medical applications

Identification of non-conserved residues essential for improving the hydrocarbon-producing activity of cyanobacterial aldehyde-deformylating oxygenase

Abstract Background Cyanobacteria produce hydrocarbons corresponding to diesel fuels by means of aldehyde-deformylating oxygenase (ADO). ADO catalyzes a difficult and unusual reaction in the conversion of aldehydes to hydrocarbons and has been widely used for biofuel production in metabolic engineering; however, its activity is low. A comparison of the amino acid sequences of highly active and less active ADOs will elucidate non-conserved residues that are essential for improving the hydrocarbon-producing activity of ADOs. Results Here, we measured the activities of ADOs from 10 representative cyanobacterial strains by expressing each of them in Escherichia coli and quantifying the hydrocarbon yield and amount of soluble ADO. We demonstrated that the activity was highest for the ADO from Synechococcus elongatus PCC 7942 (7942ADO). In contrast, the ADO from Gloeobacter violaceus PCC 7421 (7421ADO) had low activity but yielded high amounts of soluble protein, resulting in a high production level of hydrocarbons. By introducing 37 single amino acid substitutions at the non-conserved residues of the less active ADO (7421ADO) to make its sequence more similar to that of the highly active ADO (7942ADO), we found 20 mutations that improved the activity of 7421ADO. In addition, 13 other mutations increased the amount of soluble ADO while maintaining more than 80% of wild-type activity. Correlation analysis showed a solubility-activity trade-off in ADO, in which activity was negatively correlated with solubility. Conclusions We succeeded in identifying non-conserved residues that are essential for improving ADO activity. Our results may be useful for generating combinatorial mutants of ADO that have both higher activity and higher amounts of the soluble protein in vivo, thereby producing higher yields of biohydrocarbons

Role of the molten globule state in protein folding

Publisher SummaryThis chapter deals with the structure of the molten globules of various globular proteins revealed by the recent experimental studies. Recent advances in experimental techniques, including hydrogen-exchange NMR, solution X-ray scattering, and protein engineering, have provided detailed pictures of the molten globules for these proteins. The molten globule state has heterogeneous structures, in which one portion of a molecule is more organized and native-like with the other portions being less organized, although the overall structure satisfies the criteria of the molten globule state (compactness, the presence of secondary structure, and the lack of rigid tertiary structure). The chapter describes how the molten globule state has been identified as the intermediate of kinetic refolding and discusses the kinetic roles of the molten globule state in protein folding. The chapter also discusses thermodynamic stability and cooperativity of the molten globule state from the viewpoint of the hierarchy of protein folding, in which the molten globule state plays a role as a junction of two levels of the hierarchy.We dedicate this review to the late Prof. Dr. Oleg Borisovich Ptitsyn, who passed away during our preparation of this article

Rapid formation of a molten globule intermediate in refolding of α-lactalbumin

Backgound: The molten globule state is an intermediate between the native and the fully unfolded states of globular proteins and is purported to be an obligatory on-pathway intermediate of protein folding. The molten globule state of α-lactalbumin has been best characterized, but two major issues have yet to be clarified. At which stage of the kinetic refolding is the molten globule state stably organized? And what is the major driving force that stabilizes the molten globule state? We address these questions in this paper.ResultsWe have investigated the refolding kinetics of α-lactalbumin using stopped-flow CD and fluorescence, acrylamide quenching and pulsed hydrogen exchange NMR techniques. A burst-phase intermediate was observed to form within 15 ms. The intermediate was characterized by pronounced, hydrogen-bonded secondary structure, exposure of hydrophobic surfaces and the absence of tertiary structure. Furthermore, the stability of the secondary structure is the same as that in the equilibrium molten globule state.ConclusionThe burst-phase intermediate in α-lactalbumin refolding is identical with the molten globule state. Two different models, the hydrophobic collapse model and the secondary-structure coalescence model, of protein folding are discussed on the basis of the present results. The importance of solvent-separated hydrophobic interactions that stabilize the molten globule state is proposed

Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.

<p>Far-ultra violet (UV) circular dichroism (CD) spectra of the wild type and mutant ADs.</p