Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.

Active E. coli heteromeric acetyl-CoA carboxylase forms polymorphic helical tubular filaments.

The Escherichia coli heteromeric acetyl-CoA carboxylase (ACC) has four subunits assumed to form an elusive catalytic complex and are involved in allosteric and transcriptional regulation. The E. coli ACC represents almost all ACCs from pathogenic bacteria making it a key antibiotic development target to fight growing antibiotic resistance. Furthermore, it is a model for cyanobacterial and plant plastid ACCs as biofuel engineering targets. Here we report the catalytic E. coli ACC complex surprisingly forms tubes rather than dispersed particles. The cryo-EM structure reveals key protein-protein interactions underpinning efficient catalysis and how transcriptional regulatory roles are masked during catalysis. Discovering the protein-protein interaction interfaces that facilitate catalysis, allosteric and transcriptional regulation provides new routes to engineering catalytic activity and new targets for drug discovery.

Paradigm shift in xylose isomerase usage: a novel scenario with distinct applications.

Isomerases are enzymes that induce physical changes in a molecule without affecting the original molecular formula. Among this class of enzymes, xylose isomerases (XIs) are the most studied to date, partly due to their extensive application in industrial processes to produce high-fructose corn sirups. In recent years, the need for sustainable initiatives has triggered efforts to improve the biobased economy through the use of renewable raw materials. In this context, D-xylose usage is crucial as it is the second-most abundant sugar in nature. The application of XIs in biotransforming xylose, enabling downstream metabolism in several microorganisms, is a smart strategy for ensuring a low-carbon footprint and producing several value-added biochemicals with broad industrial applications such as in the food, cosmetics, pharmaceutical, and polymer industries. Considering recent advancements that have expanded the range of applications of XIs, this review provides a comprehensive and concise overview of XIs, from their primary sources to the biochemical and structural features that influence their mechanisms of action. This comprehensive review may help address the challenges involved in XI applications in different industries and facilitate the exploitation of xylose bioprocesses.

The prediction of impacted versus spontaneously erupted mandibular third molars.

BACKGROUND: To evaluate the predictive capacity of orthodontists and oral maxillofacial surgeons (OMFSs) in anticipating the process of impaction or eruption of lower third molars (L3Ms) through the examination of serial panoramic radiographs. METHODS: Sixty-eight lower third molars (L3Ms) were analyzed in 34 orthodontically treated patients without extraction. Twenty-seven OMFSs and 27 orthodontists were randomized in order to analyze the radiographs. Initially, the evaluators issued the prognosis for the L3Ms in XR1, a posterior for the XR1 + XR2. Concordance of the diagnosis was examined using Kappa statistics, and the differences between the groups of evaluators were examined using the chi-square test at p<0.05. RESULTS: When examining XR1 in cases where the teeth erupted spontaneously, the prognostic accuracy rate for OMFSs and orthodontists was similar, 63 and 65.7%, respectively (p=0.19). When evaluating XR1 + XR2, the accuracy among orthodontists (60%) was similar to that reported for XR1 (p=0.19), while OMFSs presented a reduction in the accuracy (55.3%, p<0.0001). When the L3Ms remained impacted, accuracy in XR1 was lower than in spontaneously erupting L3Ms, although similar between OMFSs (50.1%) and orthodontists (49.1%). Furthermore, for impacted L3Ms, when examining XR1 + XR2, the OMFSs presented a significant higher accuracy (71.8%, p <0.0001). CONCLUSIONS: Orthodontists and OMFSs seem unable to predict spontaneous eruption or impaction of the lower third molars from single or longitudinal x-rays. When adding a second longitudinal x-ray, orthodontists and more significantly OMFSs tend to indicate more extractions.

Electrostatic interaction optimization improves catalytic rates and thermotolerance on xylanases.

Understanding the aspects that contribute to improving proteins' biochemical properties is of high relevance for protein engineering. Properties such as the catalytic rate, thermal stability, and thermal resistance are crucial for applying enzymes in the industry. Different interactions can influence those biochemical properties of an enzyme. Among them, the surface charge-charge interactions have been a target of particular attention. In this study, we employ the Tanford-Kirkwood solvent accessibility model using the Monte Carlo algorithm (TKSA-MC) to predict possible interactions that could improve stability and catalytic rate of a WT xylanase (XynAWT) and its M6 xylanase (XynAM6) mutant. The modeling prediction indicates that mutating from a lysine in position 99 to a glutamic acid (K99E) favors the native state stabilization in both xylanases. Our lab results showed that mutated xylanases had their thermotolerance and catalytic rate increased, which conferred higher processivity of delignified sugarcane bagasse. The TKSA-MC approach employed here is presented as an efficient computational-based design strategy that can be applied to improve the thermal resistance of enzymes with industrial and biotechnological applications.

Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

Crystal structure of a novel xylose isomerase from Streptomyces sp. F-1 revealed the presence of unique features that differ from conventional classes.

BACKGROUND: Enzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation. METHODS: We applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylA1F1 and XylA2F1, from this strain. RESULTS: We showed that while XylA1F1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes. CONCLUSIONS: We have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface. GENERAL SIGNIFICANCE: Our findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass.

Bioethanol production by recycled Scheffersomyces stipitis in sequential batch fermentations with high cell density using xylose and glucose mixture.

Here, it is shown three-step investigative procedures aiming to improve pentose-rich fermentations performance, involving a simple system for elevated mass production by Scheffersomyces stipitis (I), cellular recycle batch fermentations (CRBFs) at high cell density using two temperature strategies (fixed at 30°C; decreasing from 30 to 26°C) (II), and a short-term adaptation action seeking to acclimatize the microorganism in xylose rich-media (III). Cellular propagation provided 0.52gdrycellweightgRS(-1), resulting in an expressive value of 45.9gdrycellweightL(-1). The yeast robustness in CRBF was proven by effective ethanol production, reaching high xylose consumption (81%) and EtOH productivity (1.53gL(-1)h(-1)). Regarding the short-term adaptation, S. stipitis strengthened its robustness, as shown by a 6-fold increase in xylose reductase (XR) activity. The short fermentation time (20h for each batch) and the fermentation kinetics for ethanol production from xylose are quite promising.

Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis.

OBJECTIVES: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein. RESULTS: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.5-6.5 and 35-40 °C, respectively. The enzyme stability increased 128-fold when immobilized with glyoxyl agarose, and the hydrolysis of pretreated sugar cane biomass increased by 15 % when a commercial enzyme cocktail was supplemented with immobilized ABNase. CONCLUSION: Pectin hydrolysis by recombinant ABNase plays a role in the effective application of enzymatic cocktails for biomass saccharification.