Labile Residues Research Articles

Investigation of Immune Responses to Oxidation, Deamidation, And Isomerization in Therapeutic Antibodies using Preclinical Immunogenicity Risk Assessment Assays

Product- and process- related critical quality attributes have the potential to impact pharmacokinetics, immunogenicity, potency, and safety of biotherapeutics. Among these critical quality attributes are chemical degradations, specifically oxidation, deamidation, and isomerization. These degradations can be induced by stressors such as light, pH, or temperature; they can also occur naturally under normal conditions. The immunogenicity risk of chemical degradations, particularly in the absence of aggregation, has not been thoroughly understood. In this study, model antibodies with known labile residues were stressed to induce each of the three chemical degradation classes. Aggregate-free and chemically modified antibody species were fractionalized and characterized, followed by testing in standardized and qualified preclinical immunogenicity risk assessment assays for dendritic cell internalization and presentation, monocyte activation, and pre-existing reactivity. Preclinical immunogenicity risk was assessed holistically in vitro based on changes in innate activation risk, CD4 T cell risk, and B cell risk compared to corresponding native antibody. The results of this study suggest an overall moderate increase in immune activation potential for the antibody with isomerization, with only slight increases observed in oxidized and deamidated antibodies. These findings could lend understanding to the immunogenicity risk of chemical degradations in therapeutic antibodies and therefore inform optimization engineering at particular labile residues and risk assessment under the Quality by Design framework.

Long‐term agricultural practice effects on carbon and nitrogen isotopes of soil organic matter fractions

AbstractUnderstanding the effects of long‐term traditional and alternative agricultural management practice effects on carbon (C) and nitrogen (N) cycling and storage within particulate organic matter (POM) and light fractions (LF) within various soil aggregate‐size classes can be illuminated by isotopic 13C/12C (δ13C) and 15N/14N (δ15N) differences. The objective of this study was to evaluate the effects of residue level, residue burning, tillage, and irrigation on δ13C and δ15N values of the bulk‐soil, macro‐ (>250 μm) and micro‐aggregate‐(53–250 μm), coarse‐ (>250 μm), and fine‐ (53–250 μm) POM, and coarse‐ and fine‐LF in the top 10 cm following 13 yr of consistent management in a wheat (Triticum aestivum L.)–soybean [Glycine max (L.) Merr.] double‐crop system on a silt‐loam soil in eastern Arkansas. Various treatment combinations affected (p < .05) δ13C values within the bulk‐soil and fine‐POM, as well as δ15N values within the bulk‐soil, macro‐aggregate, coarse‐LF, and fine‐LF fractions. Averaged across all other field treatments, macro‐aggregate δ15N was greater (p < .01) in the no‐tillage (NT)‐low‐ (3.23%) compared with NT–high‐residue (3.05%) and CT‐high‐ and low‐residue combination, which did not differ and averaged 3.11%, indicating that more labile residue can be achieved in the NT–high‐residue treatment combination. Results showed significant variations in aggregate‐associated δ13C and δ15N, as affected by long‐term residue and water management practices that would otherwise not have been evident from simple, bulk‐soil analysis or a short‐term field study.

Quality of fresh plant residue affects sequestration of residue derived organic material by humic acid

Increasing the retention and sequestration of plant residue carbon in agricultural soils by incorporating humic acid is the main focus of this study. This study aims to examine the effect of humic acid addition on the decomposition of plant residues of varying degrees of lability. Respiration experiments were conducted to estimate the ability of humic acid to protect plant derived organic compounds from decomposition. Humic acid reduced mineralization from all added residues and this protection effect followed the lability of the residues: vetch> wheat> oak. This could be attributed to the chemical interaction between different plant-derived organic compounds and the humic acid. Lysine was strongly adsorbed to humic acid and mineralization was reduced by 23% as a result of the strong electrostatic interaction. Applying humic acid with vetch reduced all microbial indices as a result of less substrate availability for miroorganisms. On the other hand, applying humic acid with wheat might have simulated the synthesis of extracellular enzymes and the co-metabolism of humic acid (brimming effect), resulting in an enhanced microbial structure toward a higher fungal population. This study suggests: (1) Applying humic acid to ecosystems that receive labile residues (such as vetch) to reduce mineralization and enhance carbon sequestration (2) Applying less labile residues (such as wheat) in combination with humic acid to recover degraded soils and enhance carbon sequestration.

Amount and incorporation of plant residue inputs modify residue stabilisation dynamics in soil organic matter fractions

Carbon sequestration in agricultural soils has been promoted as a means to reduce atmospheric concentrations of greenhouse gases (GHG) whilst improving soil productivity. Although there is broad agreement on practices that increase carbon (C) stocks, uncertainty remains on how agricultural management affects the stability of these gains. The fate of above-ground residue into soil organic matter (SOM) was tracked using isotopically labelled (13C and 15N) residue over 12 months in a pasture soil in sub-tropical Australia. Agricultural residue management was simulated by (1) altering the rate of residue input and (2) incorporating residue with topsoil or leaving on soil surface. Increased input and incorporation of residue increased residue-derived SOM content, with the majority of residue-derived SOM accumulating as particulate organic matter (POM) (65%) with more modest gains in mineral-associated fractions. Rapid accumulation of residue-derived SOM in the mineral-associated fractions in the initial stages of decomposition, coinciding with a high loss of labile residue components, indicate an important role for soluble OM inputs in providing an immediate and long-term sink for C and N. However, this must be considered alongside high rates of accumulation in the more readily mineralised POM fraction, particularly when a soil is approaching saturation, which is likely to lead to greater mineralisation of SOM.

The Use of a 2,2'-Azobis (2-Amidinopropane) Dihydrochloride Stress Model as an Indicator of Oxidation Susceptibility for Monoclonal Antibodies

Protein oxidation is a major pathway for degradation of biologic drug products. Past literature reports have suggested that 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator that produces alkoxyl and alkyl peroxyl radicals, is a useful model reagent stress for assessing the oxidative susceptibility of proteins. Here, we expand the applications of the AAPH model by pairing it with a rapid peptide map method to enable site-specific studies of oxidative susceptibility of monoclonal antibodies and their derivatives for comparison between formats, the evaluation of formulation components, and comparisons across the stress models. Comparing the free radical–induced oxidation model by AAPH with a light-induced oxidation model suggests that light-sensitive residues represent a subset of AAPH-sensitive residues and therefore AAPH can be used as a preliminary screen to highlight molecules that need further assessment by light models. In sum, these studies demonstrate that AAPH stress can be used in multiple ways to evaluate labile residues and oxidation sensitivity as it pertains to developability and manufacturability.

Pore characteristics regulate priming and fate of carbon from plant residue

Decomposition of the plant residue added to soil affects fate of native soil organic matter (SOM) via magnitude and direction of priming effect (PE). Soil pore characteristics, namely, pore size distribution (PSD), regulate air and liquid fluxes, as well as transport of solubilized decomposing residue to microorganisms. The goal of this study was to assess the effect of PSD on plant residue decomposition and PE as modulated by i) residue quality, ii) soil moisture status, and iii) long-term management history. We combined labeling and imaging approaches to visualize loss of decomposing residue and link it to PE in soils with two contrasting PSDs, dominated by small (5–10 μm) pores and by a combination of very small (<5 μm) and large (>30 μm) pores, respectively. The microcosms were incubated with 13C-labeled corn and soybean leaves, in soils from long-term conventional and biologically based managements. X-ray computed micro-tomography scanning was utilized to visualize loss of intact leaves at early stages (7, 14, 24 d) of decomposition. We found that PSD of soil adjacent to the decomposing plant residue played a major role in the fate of the residue and of its decomposition products. In microcosms with prevalence of small (5–10 μm) pores decomposition of corn leaves was slower, movement of decomposition products into the adjacent soil was greater, and proportion of CO2 that originated from the residue was lower than in the microcosms with prevalence of large pores. Greater positive PE took place in microcosms with small than with large pores. While these tendencies were observed in all studied soil moisture levels, management practices, and plant residue substrates, they were most pronounced in microcosms with more labile residue (soybean) in the soil from long-term biologically based management. Across treatments, the intensity of PE was greater for soils under conventional than biologically-based management. The findings emphasize importance of accounting for soil PSD when assessing processes of soil C accrual and priming.

Quantitative Proteomics Based on Optimized Data-Independent Acquisition in Plasma Analysis.

The advent of high-resolution and frequency mass spectrometry has ushered in an era of data-independent acquisition (DIA). This approach affords enormous multiplexing capacity and is particularly suitable for clinical biomarker studies. However, DIA-based quantification of clinical plasma samples is a daunting task due to the high complexity of clinical plasma samples, the diversity of peptides within the samples, and the high biologic dynamic range of plasma proteins. Here we applied DIA methodology, including a highly reproducible sample preparation and LC-MS/MS analysis, and assessed its utility for clinical plasma biomarker detection. A pancreatic cancer-relevant plasma spectral library was constructed consisting of over 14 000 confidently identified peptides derived from over 2300 plasma proteins. Using a nonhuman protein as the internal standard, various empirical parameters were explored to maximize the reliability and reproducibility of the DIA quantification. The DIA parameters were optimized based on the quantification cycle times and fragmentation profile complexity. Higher analytical and biological reproducibility was recorded for the tryptic peptides without labile residues and missed cleavages. Quantification reliability was developed for the peptides identified within a consistent retention time and signal intensity. Linear analytical dynamic range and the lower limit of quantification were assessed, suggesting the critical role of sample complexity in optimizing DIA settings. Technical validation of the assay using a cohort of clinical plasma indicated the robustness and unique advantage for targeted analysis of clinical plasma samples in the context of biomarker development.

Biomass Decomposition and Phosphorus Release from Residues of Cover Crops under No‐Tillage

Residue decomposition and P release was studied under no tillage field conditions. Cover crop residues releases significant quantities of P for the subsequent cash crop. Phosphorus release from residues is affected by precipitation regime and residue quality. A positive tradeoff was observed between crop residue P dynamics and soil coverage. Under no‐tillage field conditions, cover crop (CC) residues remain on the soil surface and decompose at a slow rate. We used three CC species (oat, Avena sativa L.; rye, Secale cereale L.; and ryegrass, Lolium multiflorum L.) to evaluate the residue biomass decomposition and P pools release–total phosphorus (Pt), inorganic phosphorus (Pi), and organic phosphorus (Po)–under no‐tillage field conditions. The dynamics of biomass and P in CC residues was evaluated through the litterbag method during two annual periods in a long‐term field experiment. Exponential decay models were fitted to each variable. Biomass decomposition and P release followed asymptotic decay models for the less labile residues (oat and rye) and single exponential decay models for the more labile ryegrass residues. Biomass and Pt showed rather equivalent dynamics, but Pi release was faster (Pi rates were three times higher and Pi plateaus were two to four times lower than biomass values). The release of Pt from CC residues reached agronomically significant quantities (2–16 kg ha−1) during the cash crop growing season and most of this Pt was released as Pi (53–100%). A positive tradeoff was observed between the release of Pi, potentially available for subsequent crop, and the residue biomass remaining over the soil. This means that Pi was released from residues, but enough biomass remained on the soil surface for erosion control. These results can be incorporated into predictive models on the contribution of CC residues to soil coverage and P nutrition of subsequent crops.

Abstract 5434: A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies

Abstract Spontaneous degradation reactions such as oxidation, glycation, deamidation, Isomerization, and racemization are non-enzymatic modifications and produce functionally damaged species reflecting aging effect at the molecular level. L-asparagine (Asn) and L-aspartate (Asp) are among the most unstable residues in proteins- linked to deamidation, isomerization and racemization Rx. Under physiological conditions, succinimide-linked deamidation of Asn occurs with t1/2as short as 6 hrs while that for isoaspartate are generally 10-fold longer. A number of biological peptides and proteins possess labile Asn and Asp residues and the formation of IsoAsp at these sites adversely affect their function. The degradation of Asn and Asp sites via succinimide pathway has significant effect on protein function. Loss of function associated with isoAsp formation are found in many biologically critical proteins and is a major source of antibody instability and micro heterogeneity, Thus it is critical to accurately determine both the levels and the site of Asn deamidation in therapeutic antibodies and proteins. Although mass Spectroscopic platforms have been at the forefront of technologies to quantitate and identify sites of isoAsp formation, it fails in accurately determining Asn level and has problems with Asp isomerization due to the various steps required for sample preparation such as denaturation, reduction, alkylation, and enzyme digestion of the Ab before analysis resulting in problems with Asp isomerization and Asn determination artifacts. Although peptide mapping is the most powerful tool in isoAsp analyses, it is very time consuming and may itself actually cause sample degradation during preparation and analysis and is not always practical when large number of samples are needed for analysis. To improve on and complement existing technologies, we have developed a bioluminescent, homogenous, robust, sensitive and easy to use assay to accurately monitor the deamidation of Asn and isomerization of aspartate. The assay is based on the use of protein isoaspartate methyltransferase to catalyze the methylation of isoaspartate using S-adenosylmethionine (SAM) and resulting in the formation of S-adenosylhomocysteine and methylated isoaspartate. The assay is very sensitive, it can detect as little as 100 fmol of IsoAsp in as little as 5-10 pmol of proteins. Citation Format: Said A. Goueli, Kevin Hsiao, Rushikesh Patel, Vishal Nashine. A high through-put bioluminescent assay to monitor the deamidation of asparagine and isomerization of aspartate residues in therapeutic proteins and antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5434. doi:10.1158/1538-7445.AM2015-5434

Fragmentation, fiber separation, decomposition, and nutrient release of secondary-forest biomass, mechanically chopped-and-mulched, and cassava production in the Amazon

No-tillage planting in mechanically-chopped secondary-forest seeks to replace slash-and-burning agriculture. We evaluated the effect of horizontal (HC) and vertical (VC) chopping-and-mulching mechanisms on vegetation fragmentation and decomposition rate and nutrient release from chopped residue, and on cassava production in eastern Amazon. Chopped-and-mulched residue was classified into four residue-size (Fs1=1–7, Fs2=7–25, Fs3=25–35, and Fs4=>35mm) and six residue-type (with husk/bark – WB, partially chopped – PC, compact – C, partially shredded into fibers – PS, completely shredded into fibers – CS, and formless residue – F) classes. In litter-bags, residual dry matter (DM) was determined at five different days after chopping-and-mulching and residue distribution on soil surface (DAD), whereas release of N, P, K, Ca, and Mg was evaluated at four days. Residues-size and -type classes showed similar decomposition behavior, with a reduction of approximately 60% of initial DM at 90 DAD. Nevertheless, reduction in DM was slow, where 52 days are necessary for half of labile residue to be decomposed, with part of labile and recalcitrant residue remaining on soil surface. DM and nutrients in residue reduced over time. DM was 25% for residues-size classes for HC, 20% for VC, and 26% for residue-type classes, on average, at 300 DAD. Nutrients remaining in residues at 300 DAD were 26% and 27% of N, 26% and 22% of P, 29% and 22% of K, 16% and 15% of Ca, and 17% and 23% of Mg, respectively for HC and VC. Release of nutrients was, generally, greater for smaller residue-size classes, similar between chopping-and-mulching mechanisms, and did not affect cassava yield.

Mass Spectrometry of Spacecraft Contamination Using Direct Analysis in Real-Time Ion Source

Spacecraft contamination was analyzed using mass spectrometry with the direct analysis in real-time ionization source. This source uses metastable helium for soft ionization and to mediate atmospheric desorption of samples into a mass spectrometer. The sampling methodology allows polymers to be assessed for the presence of vacuum labile components. Vacuum labile residues are significant sources of contamination on spacecraft optics, science instruments, and thermal control surfaces. The methodology also provides sensitive analysis of molecular contamination on spacecraft surfaces using existing spacecraft sampling procedures. This provides identification information for a wide range of molecular components including biomarker compounds.

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design

Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild-type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins.

An alkaline active xylanase: Insights into mechanisms of high pH catalytic adaptation

The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2 12 12 1 and the structure was determined at a resolution of 2.1 Å. The enzyme has the common eight-fold TIM-barrel structure of family 10 xylanases; however, unlike non-alkaline active xylanases, it has a highly negatively charged surface and a deeper active site cleft. Mutational analysis of non-conserved amino acids which are close to the acid/base residue has shown that Val169, Ile170 and Asp171 are important to hydrolyze xylan at high pH. Unlike the wild type xylanase which has optimum pH at 9–9.5, the triple mutant xylanase (V169A, I170F and D171N), which was constructed using sequence information of alkaline sensitive xylanses was optimally active around pH 7. Compared to non-alkaline active xylanases, the alkaline active xylanases have highly acidic surfaces and fewer solvent exposed alkali labile residues. Based on these results obtained from sequence, structural and mutational analysis, the possible mechanisms of high pH stability and catalysis are discussed. This will provide useful information to understand the mechanism of high pH adaptation and engineering of enzymes that can be operationally stable at high pH.

Localization of influenza virus sialoreceptors in equine respiratory tract.

This study was performed to identify the equine respiratory tract areas which express the specific receptor for equine influenza virus; findings may be useful to provide new ways to treat the infectious disease. The present work aims to visualize in situ the presence of sialoderivatives in the horse respiratory tract in order to localize sialoderivatives acting as influenza virus receptors. To this purpose, nasal mucosae, trachea, bronchus and lung parenchyma were removed from 8 mature horses of both sexes. We performed sialic acid characterization by means of mild and strong periodate oxidation and saponification, combined with lectin histochemistry and sialidase digestion, in addition to the direct evidentiation of sialic acid residues. No differences were shown between sexes. Sialic acid residues are present in the nasal mucous cell secretion, where they are linked to galactose by means of alpha2-3 linkage and are mainly C9 acetylated, and in the nasal and tracheal epithelial lining, where they are represented by periodate labile residues (alpha2-3)- and/or (alpha2-6)- linked to galactose. Specific receptors for equine influenza viruses are present at the nasal and tracheal epithelial lining cell coat levels, and in some trachea epithelial cells, but the horse possesses a preventive defence, which consists of the secretion of a mucous layer at nasal level, which could specifically inactivate the hemagglutinins of equine influenza virus; in addition, it expresses other sialoreceptors which can mask the influenza specific ones.

Use of p-nitrobenzyloxycarbonyl ( pNZ) as a permanent protecting group in the synthesis of Kahalalide F analogs

p-Nitrobenzyloxycarbonyl ( pNZ) is used for the permanent protection of ornithine in the synthesis of derivatives of the anti-tumor cyclodepsipeptide Kahalalide F that contain acid labile residues.

Investigation of neutral loss during collision-induced dissociation of peptide ions.

MS/MS fragmentation of peptides is dominated by overlapping b and y ion series. However, alternative fragmentation possibilities exist, including neutral loss. A database was generated containing 8400 MS/MS spectra of tryptic peptides assigned with high probability to an amino acid sequence (true positives) and a set of certified false (true negative) assignments for analysis of the amino terminus. A similar database was created for analysis of neutral loss at the carboxy termini using a data set of chymotryptic peptides. The analysis demonstrated that the presence of an internal basic residue, limiting proton mobility, has a profound effect on neutral loss. Peptides with fully mobile protons demonstrated minimal neutral loss, with the exception of amide bonds with proline on the carboxy terminal side, which created an intense neutral loss peak. In contrast, peptides with partial proton mobility contained many amino acids on either side of the amide bond associated with a strong neutral loss peak. Most notable among these was proline on the carboxy terminal side of an amide bond and aspartic acid on the amino terminal side of a bond. All results were found to be consistent for doubly and triply charged peptides and after adjustment for pairings across the amide bonds with particularly labile residues. The carboxy terminal of chymotryptic peptides also demonstrated significant neutral loss events associated with numerous amino acid residues. Clarification of the rules that govern neutral loss, when incorporated into analysis software, will improve our ability to correctly assign spectra to peptide sequences.

Deamidations in Recombinant Human Phenylalanine Hydroxylase: IDENTIFICATION OF LABILE ASPARAGINE RESIDUES AND FUNCTIONAL CHARACTERIZATION OF ASN → ASP MUTANT FORMS

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.

Preliminary Characterization and Crystal Structure of a Thermostable Cytochrome P450 from Thermus thermophilus

The second structure of a thermophile cytochrome P450, CYP175A1 from the thermophilic bacterium Thermus thermophilus HB27, has been solved to 1.8-A resolution. The overall P450 structure remains conserved despite the low sequence identity between the various P450s. The CYP175A1 structure lacks the large aromatic network found in the only other thermostable P450, CYP119, thought to contribute to thermal stability. The primary difference between CYP175A1 and its mesophile counterparts is the investment of charged residues into salt-link networks at the expense of single charge-charge interactions. Additional factors involved in the thermal stability increase are a decrease in the overall size, especially shortening of loops and connecting regions, and a decrease in the number of labile residues such as Asn, Gln, and Cys.

The Crystal Structure of the Allosteric Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from the Hyperthermophilic Archaeum Thermoproteus tenax

The NAD(+)-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from the hyperthermophilic archaeum Thermoproteus tenax represents an archaeal member of the diverse superfamily of aldehyde dehydrogenases (ALDHs). GAPN catalyzes the irreversible oxidation of d-glyceraldehyde 3-phosphate to 3-phosphoglycerate. In this study, we present the crystal structure of GAPN in complex with its natural inhibitor NADP(+) determined by multiple anomalous diffraction methods. The structure was refined to a resolution of 2.4 A with an R-factor of 0.21. The overall fold of GAPN is similar to the structures of ALDHs described previously, consisting of three domains: a nucleotide-binding domain, a catalytic domain, and an oligomerization domain. Local differences in the active site are responsible for substrate specificity. The inhibitor NADP(+) binds at an equivalent site to the cosubstrate-binding site of other ALDHs and blocks the enzyme in its inactive state, possibly preventing the transition to the active conformation. Structural comparison between GAPN from the hyperthermophilic T. tenax and homologs of mesophilic organisms establishes several characteristics of thermostabilization. These include protection against heat-induced covalent modifications by reducing and stabilizing labile residues, a decrease in number and volume of empty cavities, an increase in beta-strand content, and a strengthening of subunit contacts by ionic and hydrophobic interactions.

Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068.

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.