ABSTRACT
Turnover numbers (kcat), which indicate an enzyme's catalytic efficiency, have a wide range of applications in fields including protein engineering and synthetic biology. Experimentally measuring the enzymes' kcat is always time-consuming. Recently, the prediction of kcat using deep learning models has mitigated this problem. However, the accuracy and robustness in kcat prediction still needs to be improved significantly, particularly when dealing with enzymes with low sequence similarity compared to those within the training dataset. Herein, we present DeepEnzyme, a cutting-edge deep learning model that combines the most recent Transformer and Graph Convolutional Network (GCN) to capture the information of both the sequence and 3D-structure of a protein. To improve the prediction accuracy, DeepEnzyme was trained by leveraging the integrated features from both sequences and 3D-structures. Consequently, DeepEnzyme exhibits remarkable robustness when processing enzymes with low sequence similarity compared to those in the training dataset by utilizing additional features from high-quality protein 3D-structures. DeepEnzyme also makes it possible to evaluate how point mutations affect the catalytic activity of the enzyme, which helps identify residue sites that are crucial for the catalytic function. In summary, DeepEnzyme represents a pioneering effort in predicting enzymes' kcat values with improved accuracy and robustness compared to previous algorithms. This advancement will significantly contribute to our comprehension of enzyme function and its evolutionary patterns across species.
Subject(s)
Deep Learning , Enzymes , Enzymes/chemistry , Enzymes/metabolism , Enzymes/genetics , Protein Conformation , Models, Molecular , Proteins/chemistry , Proteins/metabolism , Computational Biology/methods , AlgorithmsABSTRACT
The essential microelement zinc is absorbed in the small intestine mainly by the zinc transporter ZIP4, a representative member of the Zrt/Irt-like protein (ZIP) family. ZIP4 is reportedly upregulated in many cancers, making it a promising oncology drug target. To date, there have been no reports on the turnover number of ZIP4, which is a crucial missing piece of information needed to better understand the transport mechanism. In this work, we used a non-radioactive zinc isotope, 70Zn, and inductively coupled plasma mass spectrometry (ICP-MS) to study human ZIP4 (hZIP4) expressed in HEK293 cells. Our data showed that 70Zn can replace the radioactive 65Zn as a tracer in kinetic evaluation of hZIP4 activity. This approach, combined with the quantification of the cell surface expression of hZIP4 using biotinylation or surface-bound antibody, allowed us to estimate the apparent turnover number of hZIP4 to be in the range of 0.08-0.2 s-1. The turnover numbers of the truncated hZIP4 variants are significantly smaller than that of the full-length hZIP4, confirming a crucial role for the extracellular domain in zinc transport. Using 64Zn and 70Zn, we measured zinc efflux during the cell-based transport assay and found that it has little effect on the zinc import analysis under these conditions. Finally, we demonstrated that use of laser ablation (LA) ICP-TOF-MS on samples applied to a solid substrate significantly increased the throughput of the transport assay. We envision that the approach reported here can be applied to the studies of metal transporters beyond the ZIP family.
ABSTRACT
The efficiency of catalysts relies on comprehending the underlying kinetics that govern their performance. Under the steady-state regime, the "rate" is referred to as the turnover frequency, where the reaction rate is first order with respect to catalysts. Here, we introduce the Maximum Kinetic Efficiency (MaxKinEff ) model, grounded in collision theory, to predict efficiency based on maximum turnover frequency, ð¤max TOF0 and maximum turnover number, ðmax TON0. The model was applied to molecular water oxidation using twenty-six transition metal catalysts from the first (3d), second (4d), and third (5d) rows. A thorough investigation reveals that [Ru(pda)(Br-py)2] (pda = 1,10-phenanthroline-2,9-dicarboxylate; Py = pyridinophane) exhibits a notable ð¤max TOF0 of 1176.87 × 10-5 s-1 due to its larger collision diameter (σð ð¶) and lower activation energy (Eð). Importantly, the trend in the computed ðmax TON0 values aligns with experimental TON, ðexperimental TON validating the model's accuracy. For instance, [Cp∗Ir(κ2-N,O)NO3] is identified by MaxKinEff as a standout performer, with the normalized maximum computed TON, ðmax TON0 resembling the experimental TON, ðexperimental TON = 2000.
ABSTRACT
Development of devices for production of H2 using light and a sustainable source of electrons may require the design of molecular systems combining a molecular catalyst and a photosensitizer. Evaluation of the efficiency of hydrogen production is commonly performed in homogeneous solution with a sacrificial electron donor and the report of the maximal turnover number vs catalyst ( T O N c a t lim ${TON_{cat}^{\lim } }$ ). This figure of merit is strongly dependent on deactivation pathways and does not by itself provide a benchmarking for catalysts. In particular, when the photosensitizer degradation is the primary source of limitation, a kinetic model, rationalizing literature data, shows that a decrease of the catalyst concentration leads to an increase of T O N c a t lim ${TON_{cat}^{\lim } }$ . It indicates that exceptionally high T O N c a t lim ${TON_{cat}^{\lim } }$ obtained at very low catalyst concentration shall not be considered as an indication of an exceptional catalytic system. We advocate for a systematic kinetic analysis in order to get a quantitative measure of the competitive pathways leading to T O N c a t lim ${TON_{cat}^{\lim } }$ values and to provide keys for performance improvement.
ABSTRACT
Photocatalysis is a contemporary research field given that the world's fossil energy resources including coal, mineral oil and natural gas are finite. The vast variety of photocatalytic systems demands for standardized protocols facilitating an objective comparison. While there are commonly accepted performance indicators such as the turnover number (TON) that are usually reported, to date there is no unified concept for the determination of TONs and the endpoint of the reaction during continuous measurements. Herein, we propose an algebraic approach using defined parameters and boundary conditions based on partial-least squares regression for generically calculating and predicting the turnover number and the endpoint of a photocatalytic experiment. Furthermore, the impact of the analysis period was evaluated with respect to the fidelity of the obtained TON, and the influence of the data point density along critical segments of the obtained fitting function is demonstrated.
ABSTRACT
We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2'-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1' and P2' residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1'-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1' residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1' amino acid residues.
Subject(s)
Exopeptidases , Staphylococcus aureus , Amino Acids/metabolism , Amino Acids/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Exopeptidases/metabolism , Exopeptidases/chemistry , Exopeptidases/genetics , Hydrophobic and Hydrophilic Interactions , Serine Endopeptidases , Staphylococcus aureus/enzymology , Substrate SpecificityABSTRACT
Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450BM3) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C14) is well coupled to O2 and NADPH consumption, but shorter chains (≤C12) are more poorly coupled. Hydroxylation of p-nitrophenoxydodecanoic acid by P450BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.
Subject(s)
NADPH-Ferrihemoprotein Reductase , Tryptophan , Oxidation-Reduction , Tryptophan/metabolism , NADP/metabolism , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/chemistry , NADPH-Ferrihemoprotein Reductase/metabolism , Cytochrome P-450 Enzyme System/metabolism , Fatty Acids/metabolism , Heme/metabolism , Tyrosine/metabolism , Bacterial Proteins/metabolismABSTRACT
Nitro aromatics have broad applications in industry, agriculture, and pharmaceutics. However, their industrial production is faced with many challenges including poor selectivity, heavy pollution and safety concerns. Nature provides multiple strategies for aromatic nitration, which opens the door for the development of green and efficient biocatalysts. Our group's efforts focused on a unique bacterial cytochrome P450 TxtE that originates from the biosynthetic pathway of phytotoxin thaxtomins, which can install a nitro group at C4 of l-Trp indole ring. TxtE is a Class I P450 and its reaction relies on a pair of redox partners ferredoxin and ferredoxin reductase for essential electron transfer. To develop TxtE as an efficient nitration biocatalyst, we created artificial self-sufficient P450 chimeras by fusing TxtE with the reductase domain of the bacterial P450BM3 (BM3R). We evaluated the catalytic performance of the chimeras with different lengths of the linker connecting TxtE and BM3R domains and identified one with a 14-amino-acid linker (TB14) to give the best activity. In addition, we demonstrated the broad substrate scope of the engineered biocatalyst by screening diverse l-Trp analogs. In this chapter, we provide a detailed procedure for the development of aromatic nitration biocatalysts, including the construction of P450 fusion chimeras, biochemical characterization, determination of catalytic parameters, and testing of enzyme-substrate scope. These protocols can be followed to engineer other P450 enzymes and illustrate the processes of biocatalytic development for the synthesis of nitro chemicals.
Subject(s)
Cytochrome P-450 Enzyme System , Ferredoxins , Ferredoxins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Biocatalysis , Amino Acids/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
A bimetallic triply fused copper(II) porphyrin complex (1) was prepared, comprising two monomeric porphyrin units linked through ß-ß, meso-meso, ß'-ß' triple covalent linkages and exhibiting remarkable catalytic activity for the electrochemical hydrogen evolution reaction in comparison to the analogous monomeric copper(II) porphyrin complex (2). Electrochemical investigations in the presence of a proton source (trifluoroacetic acid) confirmed that the catalytic activity of the fused metalloporphyrin occurred at a significantly lower overpotential (≈320â mV) compared to the non-fused monomer. Controlled potential electrolysis combined with kinetic analysis of catalysts 1 and 2 confirmed production of hydrogen, with 96 and 71 % faradaic efficiencies and turnover numbers of 102 and 18, respectively, with an observed rate constant of around 107 â s-1 for the dicopper complex. The results thus firmly establish triply fused porphyrin ligands as outstanding candidates for generating highly stable and efficient molecular electrocatalysts in combination with earth-abundant 3d transition metals.
Subject(s)
Metalloporphyrins , Porphyrins , Porphyrins/chemistry , Copper/chemistry , Hydrogen , Kinetics , Metalloporphyrins/chemistryABSTRACT
Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.
Subject(s)
Bacillus megaterium , Tryptophan , Tryptophan/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Oxidation-Reduction , Heme/metabolism , Tyrosine/metabolism , NADPH-Ferrihemoprotein Reductase/chemistry , Bacterial Proteins/metabolism , Bacillus megaterium/genetics , Bacillus megaterium/metabolismABSTRACT
Objective: We aim to quantify the absolute protein expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in various cells and tissues to determine the relative contribution of COX-1 and COX-2 to PGE2 production. Methods: An LC-MS method was developed and validated, then used for quantifying the absolute amounts of COX-1 and COX-2 in recombinant human COX-1 and COX-2, lysates from different cells, tissue microsomes of rodents and humans, Pirc rat colonic polyps, and biopsy specimens from squamous cell carcinoma (SCC) patients. The COX-1 and COX-2 turnover numbers were subsequently calculated based on apparent formation rates of PGE2. Results: A robust LC-MS method for quantification of COX-1 and COX-2 was developed and validated and then used to calculate their apparent turnover numbers. The results showed that COX-1 expression levels were much higher than that of COX-2 in all the tested tissues including the colonic epithelium of F344 (28-fold) and Pirc rats (20-fold), colonic polyps of Pirc rats (8-fold), and biopsy specimens of SCC patients (11-17-fold). In addition, both COX-1 and COX-2 were higher in polyps when compared to adjacent mucosa of Pirc rats. The turnover number of recombinant human COX-2 was 14-fold higher than that of recombinant human COX-1. LPS stimulation increased COX-2 protein expression in three cell lines (Raw 264.7, SCC9 and EOMA) as expected but unexpectedly increased COX-1 protein expression (13.8-fold) in EOMA cells. Conclusion: In human oral cancer tissues and cells as well as Pirc rat colon, COX-1 plays an unexpectedly but more important role than COX-2 in abnormal PGE2 production since COX-1 expression is much higher than COX-2. In addition, COX-1 expression levels are inducible in cells, and higher in polyps than surrounding mucosa in Pirc rat colon. These results indicate that targeted suppression of local COX-1 should be considered to reduce colon-specific PGE2-mediated inflammation.
ABSTRACT
Over the past 15 years, many articles have considered "nanozymes" as ferromagnetic nanoparticles having an "intrinsic peroxidase-like activity" in the presence of hydrogen peroxide. However, the definition and the catalytic activity of these nanozymes have been questioned. The present Perspective reports the main criteria that are essential to classify a nanoparticle as a nanozyme. It is important to consider that not all nanoparticles able to generate hydroxyl radicals in the presence of hydrogen peroxide without catalytic activity can be registered as nanozymes.
Subject(s)
Hydrogen Peroxide , Nanoparticles , CatalysisABSTRACT
The assessment of the operational stability of biocatalysts by conventional direct determination of the total turnover number (TTN), a useful indicator of lifetime biocatalyst productivity, via continuous isothermal experiments tends to be time-consuming, material-intensive, and prone to disturbances, especially in case of rather stable catalysts. In the present work, we present and validate two alternative methods for estimating the TTN of a biocatalyst for any desired operating temperature. The first method is a mechanistic approach, built upon mathematical derivation of enzyme deactivation models derived from first principles, in which TTN can be calculated from two straightforward isothermal biochemical batch measurements. The second method relies on a few non-isothermal, continuous-mode experiments in conjunction with mathematical modeling to determine the intrinsic deactivation parameters of the biocatalyst. We verify both methods on the test case of TEM-1 ß-lactamase-catalyzed penicillin G (Pen G) hydrolysis. Both alternative methods provide estimates of TTN which are typically within a factor of two to five or less of the values measured directly via lengthy, costly, and error-prone conventional isothermal aging tests. Therefore, both the mechanistic approach and the non-isothermal continuous approach are extremely valuable tools to enable calculation of catalyst cost contribution in continuous processing and to eliminate underperforming candidates in search of the most stable biocatalyst.
Subject(s)
Models, Theoretical , Catalysis , Hydrolysis , Kinetics , TemperatureABSTRACT
As synthetic biology enters the era of quantitative biology, mathematical information such as kinetic parameters of enzymes can offer us an accurate knowledge of metabolism and growth of cells, and further guidance on precision metabolic engineering. k cat , termed the turnover number, is a basic parameter of enzymes that describes the maximum number of substrates converted to products each active site per unit time. It reflects enzyme activity and is essential for quantitative understanding of biosystems. Usually, the k cat values are measured in vitro, thus may not be able to reflect the enzyme activity in vivo. In this case, Davidi et al. defined a surrogate k m a x v i v o (k app ) for k cat and developed a high throughput method to acquire k m a x v i v o from omics data. Heckmann et al. and Chen et al. proved that the surrogate parameter can be a good embodiment of the physiological state of enzymes and exhibit superior performance for enzyme-constrained metabolic model to the default one. These breakthroughs will fuel the development of system and synthetic biology.
ABSTRACT
In the category of rare inherited genetic disorders, phenylketonuria is a prominent example. Here, the defective phenylalanine hydroxylase enzyme fails to catalyze conversion of phenylalanine to tyrosine. This leads to not only excess deposition of phenylalanine leading to phenylalanine toxicity but also precludes the production of important glutamatergic and cholinergic neurotransmitters, leading to epileptic disorders, microcephaly, low intelligence quotient etc. For long, specialized food products are considered as preferred solution to prevent disease outcome. Different medical diets are developed for managing phenylketonuria includes amino acid mixtures, protein hydrolysates, cofactor-based therapy, large neutral amino acids and glycomacropeptides. However, despite the advent of alternate forms of diet products, the central form of treatment has still been free amino acid mixture. The formulated diet is by and large expensive and in-depth evaluation of several factors which contribute to the expense of medicated diet is requisite to create effective yet affordable avenues for management of disease. For this, we have discussed the role of various factors involved in increasing price of medicated diet and presented possible solutions to it. We have also extensively reviewed prevalence of disease, commercial diet for PKU patients, and their associated limitations. Overall, this is the first attempt to present a holistic view of balance between the overall impact of diet associated therapy and weighing it against the associated finances incurred.
ABSTRACT
Herein, green and non-toxic bismuth sulphide@graphitic carbon nitride (Bi2S3@g-C3N4) nanosheets (NCs) were firstly synthesized by ultrasonicated-assisted method and characterized with different tool. Bi2S3@g-C3N4 NCs antimicrobial activity tested against three types of microbes. As well the heterostructured Bi2S3@g-C3N4 NCs was investigated for removing dye and hexavalent chromium under visible light and showed high efficiency of photocatalytic oxidation/reduction higher than g-C3N4 alone, attributing to lower recombination photogenerated electron-hole pairs. Bi2S3@g-C3N4 NCs showed high antimicrobial efficiencies against Staphylococcus aureus (S. aureus) as a Gram positive bacterium, Escherichia coli (E. Coli)as a Gram negative bacterium and Candida albicans (C. albicans) and that the disinfection rates are 99.97%, 99.98% and 99.92%, respectively. The core mechanism is that the bacterial membrane could be destroyed by reactive oxygen species. The Bi2S3@g-C3N4 NCs is promising for environmental disinfection including water and public facilities disinfection and solar photocatalytic depollution. Turnover number (TON) and Turnover frequency (TOF) are used as concise assessment indicator for photocatalytic efficiency.
Subject(s)
Anti-Infective Agents , Staphylococcus aureus , Catalysis , Disinfection , Escherichia coliABSTRACT
Turnover numbers (kcat values) quantitatively represent the activity of enzymes, which are mostly measured in vitro. While a few studies have reported in vivo catalytic rates (kapp values) in bacteria, a large-scale estimation of kapp in eukaryotes is lacking. Here, we estimated kapp of the yeast Saccharomyces cerevisiae under diverse conditions. By comparing the maximum kapp across conditions with in vitro kcat we found a weak correlation in log scale of R2 = 0.28, which is lower than for Escherichia coli (R2 = 0.62). The weak correlation is caused by the fact that many in vitro kcat values were measured for enzymes obtained through heterologous expression. Removal of these enzymes improved the correlation to R2 = 0.41 but still not as good as for E. coli, suggesting considerable deviations between in vitro and in vivo enzyme activities in yeast. By parameterizing an enzyme-constrained metabolic model with our kapp dataset we observed better performance than the default model with in vitro kcat in predicting proteomics data, demonstrating the strength of using the dataset generated here.
Subject(s)
Enzymes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Carbon/metabolism , Databases, Factual , Genome, Fungal , Models, Biological , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolismABSTRACT
Spatially distributed populations with two sexes may face the problem that males and females concentrate in different parts of the habitat and mating and reproduction does not happen sufficiently often for the population to persist. For simplicity, to explore the impact of sex-dependent dispersal on population survival, we consider a discrete-time model for a semelparous population where individuals reproduce only once in their life-time, during a very short reproduction season. The dispersal of females and males is modeled by Feller kernels and the mating by a homogeneous pair formation function. The spectral radius of a homogeneous operator is established as basic reproduction number of the population, [Formula: see text]. If [Formula: see text], the extinction state is locally stable, and if [Formula: see text] the population shows various degrees of persistence that depend on the irreducibility properties of the dispersal kernels. Special cases exhibit how sex-biased dispersal affects the persistence of the population.
Subject(s)
Ecosystem , Models, Biological , Basic Reproduction Number , Female , Humans , Male , Population Dynamics , ReproductionABSTRACT
BACKGROUND: D-Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. D-Allulose 3-epimerase family enzymes catalyze biosynthesis of D-allulose via epimerization of D-fructose. RESULTS: A novel D-allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0-11.0. DaeB was able to catalyze D-fructose to D-allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number (kcat 367 s- 1). The coupling of DaeB treatment and yeast fermentation of 700 g L- 1 D-fructose solution yielded approximately 200 g L- 1 D-allulose, and 214 g L- 1 ethanol. CONCLUSIONS: The novel D-allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of D-allulose.
Subject(s)
Bacillus/enzymology , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/metabolism , Fructose/biosynthesis , Bacillus/genetics , Biocatalysis , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , Enzyme Stability , Ethanol/metabolism , Fermentation , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Phylogeny , Protein Structure, Secondary , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.