Biochimica et biophysica acta. Proteins and proteomics最新文献

{"title":"Reactive architecture profiling with a methyl acyl phosphate electrophile","authors":"Laura C. McGary ,&nbsp;Gemma L. Regan ,&nbsp;Stephen L. Bearne","doi":"10.1016/j.bbapap.2023.140945","DOIUrl":"10.1016/j.bbapap.2023.140945","url":null,"abstract":"<div><p><span><span>Activity-based protein profiling has facilitated the study of the activity of enzymes in </span>proteomes<span>, inhibitor development, and identification of enzymes that share mechanistic and active-site architectural features. Since methyl acyl phosphate monoesters<span> act as electrostatically selective anionic electrophiles for the covalent modification of nucleophiles that reside adjacent to cationic sites in proteins, we synthesized methyl hex-5-ynoyl phosphate (MHP) to broadly target such protein architectures. After treating the soluble proteome of </span></span></span><em>Paucimonas lemoignei</em><span><span> with MHP, biotinylating the resulting acylated proteins using click chemistry, enriching the protein adducts using streptavidin<span>, and analyzing the proteins by LC-MS/MS, a set of 240 enzymes and 132 non-enzyme proteins were identified for a wide spectrum of biological processes and from all 7 enzyme classes. Among those enzymes identified, β-hydroxybutyrate </span></span>dehydrogenase (</span><em>Pl</em><span>HBDH) and CTP synthase (</span><em>E. coli</em> orthologue, <em>Ec</em><span>CTPS) were purified as recombinant enzymes and their rates of inactivation and sites of modification by MHP and methyl acetyl phosphate (MAP) were characterized. MHP reacted more slowly with these proteins than MAP but exhibited greater specificity, despite its lack of multiple binding determinants. Generally, MAP modified more surface residues than MHP. MHP specifically modified Ser 146, Lys 156, and Lys 163 at the active site of </span><em>Pl</em>HBDH. MHP and MAP modified numerous residues of <em>Ec</em><span>CTPS with CTP furnishing the greatest level of protection against MHP- and MAP-dependent modification and inactivation, respectively, followed by ATP and glutamine. Overall, MHP served as an effective probe to identify proteins that are potentially amenable to inhibition by methyl acyl phosphates.</span></p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41189711","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Distinct dynamical features of plasmodial and human HSP70-HSP110 highlight the divergence in their chaperone-assisted protein folding","authors":"Aradhya Tripathi ,&nbsp;Sara Del Galdo ,&nbsp;Balasubramanian Chandramouli ,&nbsp;Niti Kumar","doi":"10.1016/j.bbapap.2023.140942","DOIUrl":"10.1016/j.bbapap.2023.140942","url":null,"abstract":"<div><p><span><span>HSP70 and its evolutionarily diverged co-chaperone HSP110, forms an important node in protein folding cascade. How these proteins maintain the aggregation-prone </span>proteome of malaria parasite in functional state remains underexplored, in contrast to its human orthologs. In this study, we have probed into conformational dynamics of plasmodial HSP70 and HSP110 through multiple </span><em>μ</em>s MD-simulations (ATP-state) and compared with their respective human counterparts. Simulations covered sampling of 3.4 and 2.8 μs for HSP70 and HSP110, respectively, for parasite and human orthologs. We provide a comprehensive description of the dynamic behaviors that characterize the systems and also introduce a parameter for quantifying protein rigidity. For HSP70, the interspecies comparison reveals enhanced flexibility in IA and IB subdomain within the conserved NBD, lesser solvent accessibility of the interdomain linker and distinct dynamics of the SBDβ of <em>Pf</em> HSP70 in comparison to <em>Hs</em> HSP70. In the case of HSP110, notable contrast in the dynamics of NBD, SBDβ and SBDα was observed between parasite and human ortholog. Although HSP70 and HSP110 are members of the same superfamily, we identified specific differences in the subdomain contacts in NBD, linker properties and interdomain movements in their human and parasite orthologs. Our study suggests that differences in conformational dynamics may translate into species-specific differences in the chaperoning activities of HSP70-HSP110 in the parasite and human, respectively. Dynamical features of <em>Pf</em><span> HSP70-HSP110 may contribute to the maintenance of proteostasis in the parasite during its intracellular survival in the host.</span></p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41189708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bothrops atrox venom: Biochemical properties and cellular phenotypes of three highly toxic classes of toxins","authors":"Leticia Lopes-de-Souza ,&nbsp;Fernanda Costal-Oliveira ,&nbsp;Carolina Rego Rodrigues ,&nbsp;Stephanie Stransky ,&nbsp;Thamyres C.S. de Assis ,&nbsp;Camila Liberato ,&nbsp;Dan Vivas-Ruiz ,&nbsp;Armando Yarleque Chocas ,&nbsp;Clara Guerra-Duarte ,&nbsp;Vania M.M. Braga ,&nbsp;Carlos Chávez-Olortegui","doi":"10.1016/j.bbapap.2023.140930","DOIUrl":"10.1016/j.bbapap.2023.140930","url":null,"abstract":"<div><p><span><span>Snake venoms<span> have a complex mixture of compounds that are conserved across species and act synergistically, triggering severe local and systemic effects. Identification of the toxin classes that are most damaging to cell </span></span>homeostasis would be a powerful approach to focus on the main activities that underpin envenomation. Here, we focus on the venom of </span><span><em>Bothrops</em><em> atrox</em></span>, snake responsible for most of the accidents in Amazon region of South America. We identified the key cytotoxic toxin fractions from <em>B. atrox</em><span><span><span> venom and mapped their biochemical properties, protein composition and cell damage. Five fractions were obtained by mass </span>exclusion chromatography and contained either a single class of </span>enzymatic activity (</span><em>i.e.</em>, L-amino acid oxidases or Hyaluronidases) or different activities co-distributed in two or more protein fractions (<em>e.g.</em><span>, Metalloproteinases, Serine Proteases, or Phospholipases A</span>₂<span><span>). Only three protein fractions reduced cell viability of primary human cells. Strikingly, such activity is accompanied by disruption of </span>cell attachment to substratum and to neighbouring cells. Such strong perturbation of morphological cell features indicates likely defects in tissue integrity </span><em>in vivo</em><span>. Mass spectrometry identified the main classes of toxins that contribute to these phenotypes. We provide here a strategy for the selection of key cytotoxic proteins for targeted investigation of their mechanism of action and potential synergism during snakebite envenomation. Our data highlights putative toxins (or combinations of) that may be the focus of future therapeutic interference.</span></p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41189654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metaviromics analysis of marine biofilm reveals a glycoside hydrolase endolysin with high specificity towards Acinetobacter baumannii","authors":"Georgios E. Premetis ,&nbsp;Nikolaos D. Georgakis ,&nbsp;Angeliki Stathi ,&nbsp;Nikolaos E. Labrou","doi":"10.1016/j.bbapap.2023.140918","DOIUrl":"10.1016/j.bbapap.2023.140918","url":null,"abstract":"<div><p>Multidrug-resistant (MDR) bacteria are a growing threat to the public health. Among them, the Gram-negative <span><em>Acinetobacter baumannii</em></span><span><span> is considered today as the most dangerous MDR pathogen. Phage-derived endolysins are </span>peptidoglycan<span> (PG) hydrolytic enzymes that can function as effective tools in the fight against MDR bacteria. In the present work, the viral diversity of a marine environmental sample (biofilm), formed near an industrial zone, was mined for the identification of a putative endolysin (</span></span><em>Ab</em><span>Lys2) that belongs to the glycoside hydrolase family 24 (GH24, EC 3.2.1.17). The coding sequence of </span><em>Ab</em>Lys2 was cloned and expressed in <em>E. coli</em><span>. The lytic activity and specificity of the recombinant enzyme were evaluated against suspensions of a range of Gram-positive and Gram-negative human pathogens using turbidity assays. </span><em>Ab</em>Lys2 displayed enhanced selectivity towards <em>A. baumannii</em><span> cells, compared to other bacteria. Kinetics analysis<span> was carried out to characterize the dependence of its lytic activity on pH and showed that the enzyme exhibits its maximal activity at pH 5.5. Thermostability analysis showed that </span></span><em>Ab</em>Lys2 displays melting temperature T_m<span> 47.1 °C. Florescence microscopy<span> and cell viability assays established that </span></span><em>Ab</em>Lys2 is active towards live cultures of <em>A. baumannii</em><span> cells with an inhibitory concentration IC</span>₅₀<span><span> 3.41 ± 0.09 μM. Molecular modeling allowed the prediction of important </span>amino acid residues involved in catalysis. The results of the present study suggest that </span><em>Ab</em><span>Lys2 provides efficient lytic and antimicrobial activity towards </span><em>A. baumannii</em> cells and therefore is a promising new antimicrobial against this pathogen.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10006397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Beta-KTx14.3, a scorpion toxin, blocks the human potassium channel KCNQ1","authors":"Gustavo Titaux-Delgado ,&nbsp;Andrea Estefanía Lopez-Giraldo ,&nbsp;Elisa Carrillo ,&nbsp;Luis Fernando Cofas-Vargas ,&nbsp;Luis Enrique Carranza ,&nbsp;Estuardo López-Vera ,&nbsp;Enrique García-Hernández ,&nbsp;Federico del Rio-Portilla","doi":"10.1016/j.bbapap.2023.140906","DOIUrl":"10.1016/j.bbapap.2023.140906","url":null,"abstract":"<div><p><span><span>Potassium channels<span> play a key role in regulating many physiological processes<span>, thus, alterations in their proper functioning can lead to the development of several diseases. Hence, the search for compounds capable of regulating the activity of these channels constitutes an intense field of investigation. Potassium </span></span></span>scorpion toxins are grouped into six subfamilies (α, β, γ, κ, δ, and λ). However, experimental structures and functional analyses of the long chain β-KTx subfamily are lacking. In this study, we recombinantly produced the toxins TcoKIK and beta-KTx14.3 present in the venom of </span><em>Tityus costatus</em> and <em>Lychas mucronatus</em><span> scorpions, respectively. The 3D structures of these β-KTx toxins were determined by nuclear magnetic resonance<span>. In both toxins, the N-terminal region is unstructured, while the C-terminal possesses the classic CSα/β motif. TcoKIK did not show any clear activity against frog Shaker and human KCNQ1 potassium channels; however, beta-KTx14.3 was able to block the KCNQ1 channel. The toxin-channel interaction mode was investigated using molecular dynamics simulations. The results showed that this toxin could form a stable network of polar-to-polar and hydrophobic interactions with KCNQ1, involving key conserved residues in both molecular partners. The discovery and characterization of a toxin capable of inhibiting KCNQ1 pave the way for the future development of novel drugs for the treatment of human diseases caused by the malfunction of this potassium channel.</span></span></p></div><div><h3>Statement of significance</h3><p>Scorpion toxins have been shown to rarely block human KCNQ1 channels, which participate in the regulation of cardiac processes. In this study, we obtained recombinant beta-KTx14.3 and TcoKIK toxins and determined their 3D structures by nuclear magnetic resonance. Electrophysiological studies and molecular dynamics models were employed to examine the interactions between these two toxins and the human KCNQ1, which is the major driver channel of cardiac repolarization<span>; beta-KTx14.3 was found to block effectively this channel. Our findings provide insights for the development of novel toxin-based drugs for the treatment of cardiac channelopathies involving KCNQ1-like channels.</span></p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9648865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites","authors":"M. Ines Burgos ,&nbsp;Sergio A. Dassie ,&nbsp;Gerardo D. Fidelio","doi":"10.1016/j.bbapap.2023.140920","DOIUrl":"10.1016/j.bbapap.2023.140920","url":null,"abstract":"<div><p><span>A novel mathematical development applied to protein ligand binding<span><span> thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. </span>Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m &gt; 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of </span></span>guanidinium chloride<span> on bovine serum albumin<span> thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.</span></span></p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9705435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification","authors":"Aline Larissa Gonçalves ,&nbsp;Paula Macedo Cunha ,&nbsp;Awana da Silva Lima ,&nbsp;Júlio César dos Santos,&nbsp;Fernando Segato","doi":"10.1016/j.bbapap.2023.140919","DOIUrl":"10.1016/j.bbapap.2023.140919","url":null,"abstract":"<div><p><span>Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, </span><span><em>Aspergillus fumigatus</em></span> var. <em>niveus</em><span><span> (AFUMN) has a wide variety of carbohydrate-active enzymes<span> (CAZymes), especially hydrolases, but a low number of </span></span>oxidative enzymes<span><span><span> in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane </span>bagasse<span> as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic </span></span>Polysaccharide<span> Monooxygenases (LPMOs). Thus, three LPMOs from the fungus </span></span></span><em>Thermothelomyces thermophilus</em> (<em>Tt</em>LPMO9D, <em>Tt</em>LPMO9H, and <em>Tt</em>LPMO9O) were selected, heterologous expressed in <span><em>Aspergillus nidulans</em></span><span>, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant </span><em>T. thermophilus</em><span> LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of </span><em>Tt</em>LPMO9D and ascorbic acid did not favor the conversion process in this study, while <em>Tt</em>LPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9650155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The effect of pH and nitrite on the haem pocket of GLB-33, a globin-coupled neuronal transmembrane receptor of Caenorhabditis elegans","authors":"Niels Van Brempt ,&nbsp;Roberta Sgammato ,&nbsp;Quinten Beirinckx ,&nbsp;Dietmar Hammerschmid ,&nbsp;Frank Sobott ,&nbsp;Sylvia Dewilde ,&nbsp;Luc Moens ,&nbsp;Wouter Herrebout ,&nbsp;Christian Johannessen ,&nbsp;Sabine Van Doorslaer","doi":"10.1016/j.bbapap.2023.140913","DOIUrl":"10.1016/j.bbapap.2023.140913","url":null,"abstract":"<div><p><span>Out of the 34 globins in </span><em><span>Caenorhabditis elegans</span></em><span>, GLB-33 is a putative globin-coupled transmembrane receptor<span><span> with a yet unknown function. The globin domain (GD) contains a particularly hydrophobic haem pocket, that rapidly oxidizes to a low-spin hydroxide-ligated haem state at physiological pH. Moreover, the GD has one of the fastest nitrite reductase activity ever reported for globins. Here, we use a combination of </span>electronic circular dichroism<span>, resonance Raman and electron paramagnetic resonance (EPR) spectroscopy with mass spectrometry to study the pH dependence of the ferric form of the recombinantly over-expressed GD in the presence and absence of nitrite. The competitive binding of nitrite and hydroxide is examined as well as nitrite-induced haem modifications at acidic pH. Comparison of the spectroscopic results with data from other haem proteins allows to deduce the important effect of Arg at position E10 in stabilization of exogenous ligands. Furthermore, continuous-wave and pulsed EPR indicate that ligation of nitrite occurs in a nitrito mode at pH 5.0 and above. At pH 4.0, an additional formation of a nitro-bound haem form is observed along with fast formation of a nitri-globin.</span></span></span></p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9646626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering of conserved residues near antibody heavy chain complementary determining region 3 (HCDR3) improves both affinity and stability","authors":"Merve Arslan ,&nbsp;Tuğçe Uluçay ,&nbsp;Seyit Kale ,&nbsp;Sibel Kalyoncu","doi":"10.1016/j.bbapap.2023.140915","DOIUrl":"10.1016/j.bbapap.2023.140915","url":null,"abstract":"<div><p><span><span><span>Affinity and stability are crucial parameters in antibody development and engineering approaches. Although improvement in both metrics is desirable, trade-offs are almost unavoidable. Heavy chain complementarity determining region 3 (HCDR3) is the best-known region for </span>antibody affinity but its impact on stability is often neglected. Here, we present a </span>mutagenesis study of conserved residues near HCDR3 to elicit the role of this region in the affinity-stability trade-off. These key residues are positioned around the conserved salt bridge between V</span>_H-K94 and V_H-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (V_H-K94:V_H-D101:V_H-D102) has an extensive impact on this loop's conformation, therefore simultaneous improvement in both affinity and stability. We find that the disruption of π-π stacking near HCDR3 (V_H<em>-</em>Y100E:V_L-Y49) at the V_H-V_L<span> interface cause an irrecoverable loss in stability even if it improves the affinity. Molecular simulations of putative rescue mutants exhibit complex and often non-additive effects. We confirm that our experimental measurements agree with the molecular dynamic simulations providing detailed insights for the spatial orientation of HCDR3. V</span>_H-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9649118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two different alanine dehydrogenases from Geobacillus kaustophilus: Their biochemical characteristics and differential expression in vegetative cells and spores","authors":"Miku Maeno ,&nbsp;Taketo Ohmori ,&nbsp;Daiki Nukada ,&nbsp;Haruhiko Sakuraba ,&nbsp;Takenori Satomura ,&nbsp;Toshihisa Ohshima","doi":"10.1016/j.bbapap.2023.140904","DOIUrl":"10.1016/j.bbapap.2023.140904","url":null,"abstract":"<div><p><span>Two putative alanine<span> dehydrogenase (AlaDH) genes (</span></span><em>GK2752</em> and <em>GK3448</em>) were found in the genome of a thermophilic spore-forming bacterium, <span><em>Geobacillus</em><em> kaustophilus.</em></span><span> The amino acid sequences<span> deduced from the two genes showed mutually high homology (71%), and the phylogenetic tree based on the amino acid sequences of the two putative AlaDHs and the homologous proteins showed that the two putative AlaDH genes (</span></span><em>GK2752</em> and <em>GK3448</em><span>) belong to different groups. Both of the recombinant gene products exhibited high NAD</span>⁺<span>-dependent AlaDH activity and were purified to homogeneity and characterized in detail. Both enzymes showed high stability against low and high pHs and high temperatures (70 °C). Kinetic analyses showed that the activities of both enzymes proceeded according to the same sequentially ordered Bi-Ter mechanism. X-ray crystallographic analysis showed the two AlaDHs to have similar homohexameric structures. Notably, GK3448-AlaDH was detected in vegetative cells of </span><em>G. kaustophilus</em> but not spores, while GK2752-AlaDH was present only in the spores. This is the first report showing the presence of two AlaDHs separately expressed in vegetative cells and spores.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9646114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}