Substrate Affinity Research Articles

The Highs and Lows of FBXW7: New Insights into Substrate Affinity in Disease and Development.

FBXW7 is a critical regulator of cell cycle, cell signaling, and development. A highly conserved F-box protein and component of the SKP1-Cullin-F-box (SCF) complex, FBXW7 functions as a recognition subunit within a Cullin-RING E3 ubiquitin ligase responsible for ubiquitinating substrate proteins and targeting them for proteasome-mediated degradation. In human cells, FBXW7 promotes degradation of a large number of substrate proteins, including many that impact disease, such as NOTCH1, Cyclin E, MYC, and BRAF. A central focus for investigation has been to understand the molecular mechanisms that allow the exquisite substrate specificity exhibited by FBXW7. Recent work has produced a clearer understanding of how FBXW7 physically interacts with both high-affinity and low-affinity substrates. We review new findings that provide insights into the consequences of "hotspot" missense mutations of FBXW7 that are found in human cancers. Finally, we discuss how the FBXW7-substrate interaction, and the kinases responsible for substrate phosphorylation, contribute to patterned protein degradation in C. elegans development.

Thermodynamic principle to enhance enzymatic activity using the substrate affinity

Understanding how to tune enzymatic activity is important not only for biotechnological applications, but also to elucidate the basic principles guiding the design and optimization of biological systems in nature. So far, the Michaelis-Menten equation has provided a fundamental framework of enzymatic activity. However, there is still no concrete guideline on how the parameters should be optimized towards higher activity. Here, we demonstrate that tuning the Michaelis-Menten constant ({K}_{m}) to the substrate concentration ([{{{{{rm{S}}}}}}]) enhances enzymatic activity. This guideline ({K}_{m}=[{{{{{rm{S}}}}}}]) was obtained mathematically by assuming that thermodynamically favorable reactions have higher rate constants, and that the total driving force is fixed. Due to the generality of these thermodynamic considerations, we propose {K}_{m}=[{{{{{rm{S}}}}}}] as a general concept to enhance enzymatic activity. Our bioinformatic analysis reveals that the {K}_{m} and in vivo substrate concentrations are consistent across a dataset of approximately 1000 enzymes, suggesting that even natural selection follows the principle {K}_{m}=[{{{{{rm{S}}}}}}].

Heterologous expression and characterization of salt-tolerant β-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-β-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-β-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-β-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45°C, respectively. rBGL_MK86 acted on the β-1,3-linked glucose dimer laminaribiose, but not β-1,4-linked or β-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of β-1,3 glucan) from Laminaria digitata than laminarin (β-1,3/β-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on β-1,6-linked glucose residues. The β-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.

Reduced nitrite accumulation at the primary nitrite maximum in the cyclonic eddies in the western North Pacific subtropical gyre.

Nitrite, an intermediate product of the oxidation of ammonia to nitrate (nitrification), accumulates in upper oceans, forming the primary nitrite maximum (PNM). Nitrite concentrations in the PNM are relatively low in the western North Pacific subtropical gyre (wNPSG), where eddies are frequent and intense. To explain these low nitrite concentrations, we investigated nitrification in cyclonic eddies in the wNPSG. We detected relatively low half-saturation constants (i.e., high substrate affinities) for ammonia and nitrite oxidation at 150 to 200 meter water depth. Eddy-induced displacement of high-affinity nitrifiers and increased substrate supply enhanced ammonia and nitrite oxidation, depleting ambient substrate concentrations in the euphotic zone. Nitrite oxidation is more strongly enhanced by the cyclonic eddies than ammonia oxidation, reducing concentrations and accelerating the turnover of nitrite in the PNM. These findings demonstrate a spatial decoupling of the two steps of nitrification in response to mesoscale processes and provide insights into physical-ecological controls on the PNM.

Enhanced Enzymatic Performance of β-Mannanase Immobilized on Calcium Alginate Beads for the Generation of Mannan Oligosaccharides.

Mannan oligosaccharides (MOSs) are excellent prebiotics that are usually obtained via the enzymatic hydrolysis of mannan. In order to reduce the cost of preparing MOSs, immobilized enzymes that demonstrate good performance, require simple preparation, and are safe, inexpensive, and reusable must be developed urgently. In this study, β-mannanase was immobilized on calcium alginate (CaAlg). Under the optimal conditions of 320 U enzyme addition, 1.6% sodium alginate, 2% CaCl2, and 1 h of immobilization time, the immobilization yield reached 68.3%. The optimum temperature and pH for the immobilized β-mannanase (Man-CaAlg) were 75 °C and 6.0, respectively. The Man-CaAlg exhibited better thermal stability, a high degree of pH stability, and less substrate affinity than free β-mannanase. The Man-CaAlg could be reused eight times and retained 70.34% of its activity; additionally, the Man-CaAlg showed 58.17% activity after 30 days of storage. A total of 7.94 mg/mL of MOSs, with 4.94 mg/mL of mannobiose and 3.00 mg/mL of mannotriose, were generated in the oligosaccharide production assay. It is believed that this convenient and safe strategy has great potential in the important field of the use of immobilized β-mannanase for the production of mannan oligosaccharides.

Label-free colorimetric sensor for Pb2+ determination using catalytic activity of MnO2 nanoflowers and elongated aptamer

In this study, a label-free aptasensor utilizing colorimetric properties was developed to detect Pb2+ with high sensitivity. The approach involved applying modified aptamer which enhanced the oxidase-mimicking activity of MnO2 nanoflowers. This innovative method provides an efficient means for monitoring Pb2+ ions without requiring any labeling techniques. The fundamental principle of this aptasensor is based on the adsorption of a modified aptamer onto MnO2 nanoflowers' surface, which in turn increases their affinity for chromogenic substrates and enhances their catalytic activity. The proposed aptasensor exploits the high sensitivity due to the extension of the aptamer sequence length by terminal deoxynucleotidyl transferase (TdT). Under optimum experimental conditions, the developed colorimetric aptasensor indicated a linear detection range from 4 to 80 nM with a limit of detection (LOD) of 1.4 nM. Moreover, the aptasensor successfully monitored Pb2+ in the drinking water, milk and human serum samples. Henceforth, the colorimetric aptasensor exhibited in this study possesses several benefits such as uncomplicated operation, cost-effectiveness, label-free detection and remarkable sensitivity. Thus rendering it a suitable option for analyzing intricate samples.

Persulfidation maintains cytosolic G6PDs activity through changing tetrameric structure and competing cysteine sulfur oxidation under salt stress in Arabidopsis and tomato.

Glucose-6-phosphate dehydrogenases (G6PDs) are essential regulators of cellular redox. Hydrogen sulfide (H2 S) is a small gasotransmitter that improves plant adaptation to stress; however, its role in regulating G6PD oligomerization to resist oxidative stress remains unknown in plants. Persulfidation of cytosolic G6PDs was analyzed by mass spectrometry (MS). The structural change model of AtG6PD6 homooligomer was built by chemical cross-linking coupled with mass spectrometry (CXMS). We isolated AtG6PD6C159A and SlG6PDCC155A transgenic lines to confirm the invivo function of persulfidated sites with the g6pd5,6 background. Persulfidation occurs at Arabidopsis G6PD6 Cystine (Cys)159 and tomato G6PDC Cys155, leading to alterations of spatial distance between lysine (K)491-K475 from 42.0 Å to 10.3 Å within the G6PD tetramer. The structural alteration occurs in the structural NADP+ binding domain, which governs the stability of G6PD homooligomer. Persulfidation enhances G6PD oligomerization, thereby increasing substrate affinity. Under high salt stress, cytosolic G6PDs activity was inhibited due to oxidative modifications. Persulfidation protects these specific sites and prevents oxidative damage. In summary, H2 S-mediated persulfidation promotes cytosolic G6PD activity by altering homotetrameric structure. The cytosolic G6PD adaptive regulation with two kinds of protein modifications at the atomic and molecular levels is critical for the cellular stress response.

Psychrophilic enzymes: strategies for cold-adaptation.

Psychrophilic organisms thriving at near-zero temperatures synthesize cold-adapted enzymes to sustain cell metabolism. These enzymes have overcome the reduced molecular kinetic energy and increased viscosity inherent to their environment and maintained high catalytic rates by development of a diverse range of structural solutions. Most commonly, they are characterized by a high flexibility coupled with an intrinsic structural instability and reduced substrate affinity. However, this paradigm for cold-adaptation is not universal as some cold-active enzymes with high stability and/or high substrate affinity and/or even an unaltered flexibility have been reported, pointing to alternative adaptation strategies. Indeed, cold-adaptation can involve any of a number of a diverse range of structural modifications, or combinations of modifications, depending on the enzyme involved, its function, structure, stability, and evolutionary history. This paper presents the challenges, properties, and adaptation strategies of these enzymes.

Arginine-depleting Enzymes, A Potential Treatment Option for Tumors With Arginine Auxotrophy : A Review

The World Health Organization reports that one of the top global causes of illness and mortality is cancer, with nearly 10 million deaths in 2020. Changes in cellular metabolism are common characteristics of a wide variety of malignancies. Enzymatic deficits cause many tumors to lose the ability to synthesize amino acids required for their growth, survival, or proliferation. Thus, some tumors depend on the extra-cellular supply of specific amino acids to meet their needs, allowing them to survive. Amino acid depletion as a targeted therapy takes advantage of these tumor traits by depleting certain amino acids in the body that is required for the tumor to survive. This review aims to discuss the potential and challenges of arginine-depleting enzymes as a means in treating arginine auxotrophic cancers. Previously, arginine deiminase (ADI) of bacterial origin has been studied for the in vivo arginine auxotrophic tumour therapy. However, it has been hampered by drawbacks, including immunogenicity and toxicity issues. Thus, human arginase I (hARGI) has been considered a better candidate due to its low mmunogenicity and toxicity effects. However, hARGI’s application as an anti-cancer drug is hindered by its low activity towards arginine owing to its high Km values indicating the enzyme’s low substrate affinity. Thus, it is necessary to improve the enzyme catalytic capability and stability for more practical application in therapeutic cancer treatment. With the advancement of bioinformatics tools, more studies are anticipated to rationally engineer the enzyme for more practical clinical application in the treatment of arginine auxotrophic cancers.

Exploring the Chain Release Mechanism from an Atypical Apicomplexan Polyketide Synthase.

Polyketide synthases (PKSs) are megaenzymes that form chemically diverse polyketides and are found within the genomes of nearly all classes of life. We recently discovered the type I PKS from the apicomplexan parasite Toxoplasma gondii, TgPKS2, which contains a unique putative chain release mechanism that includes ketosynthase (KS) and thioester reductase (TR) domains. Our bioinformatic analysis of the thioester reductase of TgPKS2, TgTR, suggests differences compared to other systems and hints at a possibly conserved release mechanism within the apicomplexan subclass Coccidia. To evaluate this release module, we first isolated TgTR and observed that it is capable of 4 electron (4e-) reduction of octanoyl-CoA to the primary alcohol, octanol, utilizing NADH. TgTR was also capable of generating octanol in the presence of octanal and NADH, but no reactions were observed when NADPH was supplied as a cofactor. To biochemically characterize the protein, we measured the catalytic efficiency of TgTR using a fluorescence assay and determined the TgTR binding affinity for cofactor and substrates using isothermal titration calorimetry (ITC). We additionally show that TgTR is capable of reducing an acyl carrier protein (ACP)-tethered substrate by liquid chromatography mass spectrometry and determine that TgTR binds to holo-TgACP4, its predicted cognate ACP, with a KD of 5.75 ± 0.77 μM. Finally, our transcriptional analysis shows that TgPKS2 is upregulated ∼4-fold in the parasite's cyst-forming bradyzoite stage compared to tachyzoites. Our study identifies features that distinguish TgPKS2 from well-characterized systems in bacteria and fungi and suggests it aids the T. gondii cyst stage.

The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming

The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit’s AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.

Alanine dehydrogenases from four different microorganisms: characterization and their application in L-alanine production

BackgroundAlanine dehydrogenase (AlaDH) belongs to oxidoreductases, and it exists in several different bacteria species and plays a key role in microbial carbon and nitrogen metabolism, spore formation and photosynthesis. In addition, AlaDH can also be applied in biosynthesis of L-alanine from cheap carbon source, such as glucose.ResultsTo achieve a better performance of L-alanine accumulation, system evaluation and comparison of different AlaDH with potential application value are essential. In this study, enzymatic properties of AlaDH from Bacillus subtilis 168 (BsAlaDH), Bacillus cereus (BcAlaDH), Mycobacterium smegmatis MC2 155 (MsAlaDH) and Geobacillus stearothermophilus (GsAlaDH) were firstly carefully investigated. Four different AlaDHs have few similarities in optimum temperature and optimum pH, while they also exhibited significant differences in enzyme activity, substrate affinity and enzymatic reaction rate. The wild E. coli BL21 with these four AlaDHs could produce 7.19 g/L, 7.81 g/L, 6.39 g/L and 6.52 g/L of L-alanine from 20 g/L glucose, respectively. To further increase the L-alanine titer, competitive pathways for L-alanine synthesis were completely blocked in E. coli. The final strain M-6 could produce 80.46 g/L of L-alanine with a yield of 1.02 g/g glucose after 63 h fed-batch fermentation, representing the highest yield for microbial L-alanine production.ConclusionsEnzyme assay, biochemical characterization and structure analysis of BsAlaDH, BcAlaDH, MsAlaDH and GsAlaDH were carried out. In addition, application potential of these four AlaDHs in L-alanine productions were explored. The strategies here can be applied for developing L-alanine producing strains with high titers.

Bioaugmentation Potential Investigation Using a Phenol Affinity Analysis of Three Acinetobacter Strains in a Multi-Carbon-Source Condition

Bioaugmentation potential and phenol substrate affinity in a multi-carbon-source condition for three Acinetobacter strains (Acinetobacter towneri CFII-87, Acinetobacter johnsonii CFII-99A and Acinetobacter sp. CFII-98) were demonstrated. First, the phenol biodegradation ability of the strains was analyzed in batch experiments with phenol as the sole carbon source. All strains degraded phenol at 100 and 500 mg·L−1 initial concentrations; the maximum specific growth rates were 0.59 and 0.30 d−1 for A. towneri CFII-87, 0.50 and 0.20 d−1 for A. johnsonii CFII-99A, and 0.64 and 0.29 d−1 for A. sp. CFII-98, respectively. For the two tested phenol concentrations, no lag phase was observed for the A. towneri CFII-87 strain, A. sp. CFII-98 presented 4 h and 8 h lag phase, while A. johnsonii CFII-99A presented 3 h and 12 h lag phases. Phenol carbon source dependency of the strains was tested in a multi-carbon-source condition (on phenol-rich synthetic wastewater), both for individual strains and for a consortium prepared as an equal mixture of the three strains. The strains A. towneri CFII-87 and A. sp. CFII-98 and the consortia degraded phenol in 16 h while there was no other significant carbon source consumption during the 48 h trial, as shown by the constant non-phenolic residual chemical oxygen demand (COD) and volatile suspended solids (VSS) concentration after the depletion of phenol. The strain A. johnsonii CFII-99A, however, consumed phenol within 24 h and a further decrease in non-phenolic COD and increase in biomass was also observed upon the depletion of phenol. The highest specific phenol removal rate of 282.11 mg phenol·g VSS∙h−1 was observed in the case of the strain A. towneri CFII-87, followed by A. sp. CFII-98, the consortium and A. johnsonii CFII-99A with 178.84, 146.76 and 141.01 mg phenol·g VSS∙h−1, respectively. Two bacterial strains (A. towneri CFII-87, A. sp. CFII-98) presented a strong affinity to phenol, utilizing it as a primary carbon source, and thus, their use in the bioaugmentation of wastewater bioreactors indicated the viable potential to increase the phenol removal rate of these systems.

Co-immobilization of bienzyme HRP/GOx on highly stable hierarchically porous MOF with enhanced catalytic activity and stability: Kinetic and thermodynamic studies

Immobilization of enzymes onto solid carriers usually results in the reduction of enzyme catalytic activity. To solve this tricky problem, we proposed a novel strategy by accurately constructing the micro-mesoporous channel structure of highly stable hierarchically porous metal-organic framework (HP-Zr-MOF) for co-immobilization of horseradish peroxidase (HRP) and glucose oxidase (GOx). Notably, the precise construction and co-regionalization of HP-Zr-MOF with hierarchical structure can efficiently suppress enzyme leaching and significantly improve the catalytic efficiency, enzyme activity and the affinity for substrates of co-immobilized bienzyme reactor (GOx&HRP@HP-Zr-MOF). The Km value for GOx&HRP@HP-Zr-MOF was 0.2050 mM, which was 0.66-fold less than that of free GOx&HRP, while the Ka value for GOx&HRP@HP-Zr-MOF was superior to that of free GOx&HRP, manifesting the affinity of GOx&HRP@HP-Zr-MOF for substrates strengthened. Importantly, the GOx&HRP@HP-Zr-MOF exhibited favorable stability and reusability, maintaining 97.4% of the original activity after 1 h incubation at 60 °C and over 90% initial activity after 30 cycles. Moreover, the GOx&HRP@HP-Zr-MOF was applied to the colorimetric detection of glucose, manifesting outstanding detection capability. Finally, the GOx&HRP@HP-Zr-MOF exhibited excellent catalytic efficiency for the degradation of 2,4-dichlorophenol, which indicated that the bio-catalytic material has potential practical application value in wastewater treatment.

Novel colorimetric detection of oxytetracycline in foods by copper nanozyme

In this study, copper nanozyme (CuNZs) possess good laccase-like activity were synthesized by grinding method with cupric chloride dihydrate as copper source, sodium borohydride as reducing agent and β-cyclodextrin as protective agent. The CuNZs can oxidize colorless 2,4-dinitrophenol (2,4-DP) to red product. When oxytetracycline (OTC) was added to the above three solutions, the color changed from red to orange and the absorbance increased again, indicating that OTC was also an affinity substrate for CuNZs. When CuNZs was mixed with OTC alone, the color changed from colorless to yellow, and the absorption intensity was related to OTC concentration. It has good selectivity and sensitivity, and had a good linear response to the concentration of OTC in the range of 50–500 μM, and the limit of detection was 0.148 μM. Thus, a fast and simple colorimetric assay for the determination of OTC was established by using the laccase-like activity of CuNZs, and it was applied successfully to detect OTC in food samples.

Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Identification of structural determinants of nicotinamide phosphoribosyl transferase (NAMPT) activity and substrate selectivity

NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD+ by sirtuins, PARPs, and other NAD+-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.

Stability and mixing behavior of vanadium-iron oxide monolayers on Pt(111) and Ru(0001) substrates

Cation mixing is a well-recognized means to obtain oxides of desired functionality with predetermined structure and stoichiometry, which yet has been only little analyzed at the nanoscale. In this context, we present a comparative analysis of the stability and mixing properties of O-poor and O-rich two-dimensional V–Fe oxides grown on Pt(111) and Ru(0001) surfaces, with the aim of gaining an insight into the role of substrate and oxygen conditions on the accessible Fe contents. We find that due to the high oxygen affinity of the Ru substrate, the mixed O-rich layers are highly stable while the stability of O-poor layers is limited to inaccessibly oxygen-poor environments. In contrast, on the Pt surface, O-poor and O-rich layers coexist with, however, a much lower Fe content in the O-rich phase. We show that cationic mixing (formation of mixed V–Fe pairs) is favored in all considered systems. It results from local cation–cation interactions, reinforced by a site effect in O-rich layers on the Ru substrate. In O-rich layers on Pt, Fe–Fe repulsion is so large that it precludes the possibility of substantial Fe content. These findings highlight the subtle interplay between structural effects, oxygen chemical potential, and substrate characteristics (work function and affinity towards oxygen), which governs the mixing of complex 2D oxide phases on metallic substrates.

Genetic variants causing G6PD deficiency: Clinical and biochemical data support new WHO classification.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in erythrocytes causes acute haemolytic anaemia upon exposure to fava beans, drugs, or infection; and it predisposes to neonatal jaundice. The polymorphism of the X-linked G6PD gene has been studied extensively: allele frequencies of up to 25% of different G6PD deficient variants are known in many populations; variants that cause chronic non-spherocytic haemolytic anaemia (CNSHA) are instead all rare. WHO recommends G6PD testing to guide 8-aminoquinolines administration to prevent relapse of Plasmodium vivax infection. From a literature review focused on polymorphic G6PD variants we have retrieved G6PD activity values of 2291 males, and for the mean residual red cell G6PD activity of 16 common variants we have obtained reliable estimates, that range from 1.9% to 33%. There is variation in different datasets: for most variants most G6PD deficient males have a G6PD activity below 30% of normal. There is a direct relationship between residual G6PD activity and substrate affinity (Km G6P ), suggesting a mechanism whereby polymorphic G6PD deficient variants do not entail CNSHA. Extensive overlap in G6PD activity values of individuals with different variants, and no clustering of mean values above or below 10% support the merger of class II and class III variants.

Environmental memory of microbes regulates the response of soil enzyme kinetics to extreme water events: Drought-rewetting-flooding

How environmental history mediates the response of microbial activities to dry-wet cycle (D/W) in monsoon regions is poorly investigated. In this study, soil from Red River Delta of Vietnam was incubated for 52 days, which included 14 days at 20% water holding capacity (WHC), followed by 14 days rewetted to 60% WHC (rewetting) or 100% WHC (flooding). The kinetics of β-glucosidase, chitinase and acid phosphomonoesterase enzymes, microbial respiration, microbial biomass C and P were measured.The activities of all soil enzymes (Vmax) decreased by 20–42% due to drought but partially or totally recovered after two weeks of rewetting or flooding. The substrate affinity (Km) of all measured enzymes were fluctuating at initial drought and then dropped down by 33–55% after 14 days prolonged drought. Chitinase was the only enzyme showing the decoupling of Vmax and Km at the onset of drought, indicating different factors regulating enzyme synthesis and substrate affinity for the N demands of microorganisms. Pulse rewetting significantly reduced catalytic efficiency (Ka) but boosted turnover time (Tt) of chitinase and β-glucosidase. Rewetting prolonged Tt as compared to flooding by the end of the experiment. Moreover, both soil basal respiration and substrate induced respiration were significantly inhibited by drought and recovered to the control level after rewetting. In conclusion, soil with history of D/W triggers the recovery of microbial activities after D/W cycles and rewetting has a great impact on enzyme systems, while flooding induces more effects on enzyme synthesis.