scholarly journals Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class

2021 ◽  
Vol 17 (9) ◽  
pp. e1009446
Author(s):  
Elzbieta Rembeza ◽  
Martin K. M. Engqvist

Only a small fraction of genes deposited to databases have been experimentally characterised. The majority of proteins have their function assigned automatically, which can result in erroneous annotations. The reliability of current annotations in public databases is largely unknown; experimental attempts to validate the accuracy within individual enzyme classes are lacking. In this study we performed an overview of functional annotations to the BRENDA enzyme database. We first applied a high-throughput experimental platform to verify functional annotations to an enzyme class of S-2-hydroxyacid oxidases (EC 1.1.3.15). We chose 122 representative sequences of the class and screened them for their predicted function. Based on the experimental results, predicted domain architecture and similarity to previously characterised S-2-hydroxyacid oxidases, we inferred that at least 78% of sequences in the enzyme class are misannotated. We experimentally confirmed four alternative activities among the misannotated sequences and showed that misannotation in the enzyme class increased over time. Finally, we performed a computational analysis of annotations to all enzyme classes in the BRENDA database, and showed that nearly 18% of all sequences are annotated to an enzyme class while sharing no similarity or domain architecture to experimentally characterised representatives. We showed that even well-studied enzyme classes of industrial relevance are affected by the problem of functional misannotation.

2020 ◽  
Author(s):  
Elzbieta Rembeza ◽  
Martin KM Engqvist

Only a small fraction of genes deposited to databases has been experimentally characterised. The majority of proteins have their function assigned automatically, which can result in erroneous annotations. The reliability of current annotations in public databases is largely unknown; experimental attempts to validate the accuracy of existing annotations are lacking. In this study we performed an overview of functional annotations to the BRENDA enzyme database. We first applied a high-throughput experimental platform to verify functional annotations to an enzyme class of S-2-hydroxyacid oxidases (EC 1.1.3.15). We chose 122 representative sequences of the class and screened them for their predicted function. Based on the experimental results, predicted domain architecture and similarity to previously characterised S-2-hydroxyacid oxidases, we inferred that at least 78% of sequences in the enzyme class are misannotated. We experimentally confirmed four alternative activities among the misannotated sequences and showed that misannotation in the enzyme class increased over time. Finally, we performed a computational analysis of annotations to all enzyme classes in BRENDA database, and showed that nearly 18% of all sequences are annotated to an enzyme class while sharing no similarity to experimentally characterised representatives. We showed that even well-studied enzyme classes of industrial relevance are affected by the problem of functional misannotation.


2016 ◽  
Vol 94 (4) ◽  
pp. 392-400 ◽  
Author(s):  
Leila Mokhtabad Amrei ◽  
Peter W. Dibble ◽  
René T. Boeré

The title compound Dipp(H)P–P(H)Dipp where Dipp = 2,6-diisopropylyphenyl has been prepared starting from DippPH2 via DippPHLi reacting 2:1 with BrCH2CH2Br. A high-resolution single-crystal X-ray diffraction study at 153 ± 2 K provides an accurate description of the structure in the solid state, only the fourth dihydro diorganophosphane to have been so characterized. The Raman spectrum of the solid shows an intense, broad peak for P–H stretching at 2314 cm−1 and two peaks at 460 and 450 cm−1 for P–P stretching, assignments confirmed by RHF/6-31G+(3d,2p) frequency calculations undertaken on the anti conformers. 31P, 31P{1H }, 1H{31P}, and 13C{1H } solution NMR spectra in CDCl3 are consistent with the presence of 58% meso and 42% rac diastereomers. Analysis of their AA′XX′ spin systems enabled calculation of 1JPP′ values of (–)189 and (–)205 Hz, respectively. A B3LYP/6-31G(d,p) computational analysis of the energetics of rotation about the P–P bond in both diastereomers shows that the anti and two gauche conformers are likely to be populated approximately equally in each. The resulting average ϕ angles (torsions of the phosphorus lone pairs) are 110° for meso and 103° for rac; the smaller ϕ angle fits with the larger 1JPP′ value for the rac isomer.


2019 ◽  
Vol 21 (15) ◽  
pp. 8073-8080 ◽  
Author(s):  
Wanlin Cai ◽  
Huize Zhang ◽  
Xi Yan ◽  
Anchong Zhao ◽  
Rongxing He ◽  
...  

The microscopic mechanism of high color purity was elucidated by computational investigation.


2004 ◽  
Vol 01 (02) ◽  
pp. 329-343
Author(s):  
A. C. BENIM ◽  
D. BRILLERT ◽  
M. CAGAN

A 3D computational analysis has been applied to investigate direct transfer, pre-swirl systems for gas turbine cooling. Alternative computational procedures have been applied and results have been compared with measurements. Based on these validation studies, strategies for modeling such systems have been proposed. Present results suggest that sufficiently accurate predictions can be obtained using a quasi-steady analysis applying the "frozen rotor" approach for treating the interface between the stationary and rotating domains.


2018 ◽  
Vol 115 (45) ◽  
pp. E10521-E10530 ◽  
Author(s):  
Simone Raugei ◽  
Lance C. Seefeldt ◽  
Brian M. Hoffman

Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N2→ 2NH3reduction involves the reductive elimination (re) of H2from two [Fe–H–Fe] bridging hydrides bound to the catalytic [7Fe–9S–Mo–C–homocitrate] FeMo-cofactor (FeMo-co). These studies rationalize the Lowe–Thorneley kinetic scheme’s proposal of mechanistically obligatory formation of one H2for each N2reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (i) This shows that to prevent spurious disruption of FeMo-co having accumulated 4[e−/H+] it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (ii) These calculations indicate an important role of sulfide hemilability in the overall conversion ofE0to a diazene-level intermediate. (iii) Perhaps most importantly, they explain (iiia) how the enzyme mechanistically couples exothermic H2formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutralre/oxidative-addition equilibrium, (iiib) while preventing the “futile” generation of two H2without N2reduction: hydrideregenerates an H2complex, but H2is only lost when displaced by N2, to form an end-on N2complex that proceeds to a diazene-level intermediate.


Author(s):  
Gerardo Diaz

Alternative fuels, mainly generated from waste, constitute a possible option to reduce the demand for fossil fuels. For instance, biogas can be obtained from cow manure using anaerobic digesters, a technology that is currently being implemented in several dairy farms. Synthesis gas, also known as syngas, is a mixture of hydrogen and carbon monoxide that may have fractions of other components such as nitrogen, carbon dioxide, and methane, depending on the gasifying method used. However, before a change to alternative fuels can be made, the performance of common appliances and industrial equipment using these fuels needs to be investigated. In this paper, a computational analysis of the performance of an air heater using natural gas, biogas, and syngas is performed. The results show that alternative fuels with heat values almost an order of magnitude lower than natural gas can be used in commercial air heaters with only minor variations in thermal performance. The main drawback is the higher flow rates required for alternative fuels, specially for syngas.


Author(s):  
Jeff Gelles

Mechanoenzymes are enzymes which use a chemical reaction to power directed movement along biological polymer. Such enzymes include the cytoskeletal motors (e.g., myosins, dyneins, and kinesins) as well as nucleic acid polymerases and helicases. A single catalytic turnover of a mechanoenzyme moves the enzyme molecule along the polymer a distance on the order of 10−9 m We have developed light microscope and digital image processing methods to detect and measure nanometer-scale motions driven by single mechanoenzyme molecules. These techniques enable one to monitor the occurrence of single reaction steps and to measure the lifetimes of reaction intermediates in individual enzyme molecules. This information can be used to elucidate reaction mechanisms and determine microscopic rate constants. Such an approach circumvents difficulties encountered in the use of traditional transient-state kinetics techniques to examine mechanoenzyme reaction mechanisms.


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