Localization of the essential histidine and carboxylate group in D-xylose isomerases.
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ABSTRACT:
SUBMITTER: Vangrysperre W
PROVIDER: S-EPMC1133690 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
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Project description:Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.
| S-EPMC1148827 | biostudies-other
Project description:D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.
| S-EPMC1138640 | biostudies-other
Project description:Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation.Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw(-1) h(-1), and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw(-1) h(-1), much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene.This study demonstrated that XIs clustered in the mammal gut Bacteroidetes group were able to be expressed functionally in S. cerevisiae and background strain anaerobic adaptive evolution in xylose medium is essential for the screening of functional XIs. The methods outlined in this paper are instructive for the identification of novel XIs that are functional in S. cerevisiae.
| S-EPMC4436767 | biostudies-literature
Project description:The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.
| S-EPMC207900 | biostudies-other
Project description:Interfacing synthetic materials with biomacromolecules provides new systems for biological applications. We report the creation of a reversible multivalent supramolecular "zipper" recognition motif between gold nanoparticles and proteins. In this assembly, carboxylate-functionalized nanoparticles interact strongly with oligohistidine tags. This interaction can be tuned through His-tag length, and offers unique binding profiles based on the pH and electrolyte concentration of the medium.
| S-EPMC4113908 | biostudies-literature
Project description:Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation.Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice).Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.
| S-EPMC5407363 | biostudies-other
Project description:The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two cis-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between beta-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression.
| S-EPMC2805319 | biostudies-literature
Project description:Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.
| S-EPMC107040 | biostudies-literature
Project description:Two-component systems (TCSs) are key elements in bacterial signal transduction in response to environmental stresses. TCSs generally consist of sensor histidine kinases (SKs) and their cognate response regulators (RRs). Many SKs exhibit autokinase, phosphoryltransferase and phosphatase activities, which regulate RR activity through a phosphorylation and dephosphorylation cycle. However, how SKs perform different enzymatic activities is poorly understood. Here, several crystal structures of the minimal catalytic region of WalK, an essential SK from Lactobacillus plantarum that shares 60% sequence identity with its homologue VicK from Streptococcus mutans, are presented. WalK adopts an asymmetrical closed structure in the presence of ATP or ADP, in which one of the CA domains is positioned close to the DHp domain, thus leading both the ?- and ?-phosphates of ATP/ADP to form hydrogen bonds to the ?- but not the ?-nitrogen of the phosphorylatable histidine in the DHp domain. In addition, the DHp domain in the ATP/ADP-bound state has a 25.7° asymmetrical helical bending coordinated with the repositioning of the CA domain; these processes are mutually exclusive and alternate in response to helicity changes that are possibly regulated by upstream signals. In the absence of ATP or ADP, however, WalK adopts a completely symmetric open structure with its DHp domain centred between two outward-reaching CA domains. In summary, these structures of WalK reveal the intrinsic dynamic properties of an SK structure as a molecular basis for multifunctionality.
| S-EPMC5633905 | biostudies-literature
Project description:Thiol isomerases are multifunctional enzymes that influence protein structure via their oxidoreductase, isomerase, and chaperone activities. These enzymes localize at high concentrations in the endoplasmic reticulum of all eukaryotic cells where they serve an essential function in folding nascent proteins. However, thiol isomerases can escape endoplasmic retention and be secreted and localized on plasma membranes. Several thiol isomerases including protein disulfide isomerase, ERp57, and ERp5 are secreted by and localize to the membranes of platelets and endothelial cells. These vascular thiol isomerases are released following vessel injury and participate in thrombus formation. Although most of the activities of vascular thiol isomerases that contribute to thrombus formation are yet to be defined at the molecular level, allosteric disulfide bonds that are modified by thiol isomerases have been described in substrates such as ?IIb?3, ?v?3, GPIb?, tissue factor, and thrombospondin. Vascular thiol isomerases also act as redox sensors. They respond to the local redox environment and influence S-nitrosylation of surface proteins on platelets and endothelial cells. Despite our rudimentary understanding of the mechanisms by which thiol isomerases control vascular function, the clinical utility of targeting them in thrombotic disorders is already being explored in clinical trials.
| S-EPMC4990854 | biostudies-literature