Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl) succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O-2-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

QM and QM/MM methods compared: case studies on reaction mechanisms of metalloenzymes

The review focus is a comparison of QM and QM/MM modeling techniques applied to study of metalloenzymes. The chapter aim is to highlight many of the advantages and potential pitfalls of the exciting and revolutionary QM/MM techniques using both large QM/MM systems and QM-only modeling as references. The review is illustrated by case studies for isopenicillin N synthase, ethylbenzene dehydrogenase, cytochrome P450 enzyme, AlkB DNA repair enzyme as well as 4-hydroxyphenylpyruvate dioxygenase. We find many advantages in various QM/MM techniques, over the more traditional QM cluster approaches, while at the same time offering some advice about how to avoid potential complications arising from some of these approaches’ most notable drawbacks. We conclude that while there will always be an important role for QM cluster models, in computational studies, the revolutionary developments in QM/MM techniques open a bright and exciting future of new research

Liquid chromatographic techniques in separation of betacyanins and their derivatives from red beet roots

A study on separation of betacyanins and their decarboxylated as well as dehydrogenated derivatives obtained from red beet roots (Beta vulgaris L.) in high pressure liquid chromatography (HPLC) and ion-pair high-speed counter-current chromatography (IP-HSCCC) was performed. The IP-HSCCC process was accomplished in the ‘head-to-tail’ mode in a solvent system composed of butanol — acetonitrile — water (5:1:6 v/v/v, acidifi ed with 0.7% trifl uoroacetic acid). The HPLC separation was performed in a typical reversed phase mode with mass spectrometry (ESI-MS) and diode-array (DAD) detection. The chromatographic profi les of betalains obtained in these two techniques were signifi cantly diff erent. In HPLC, the most polar compounds, like betanin and isobetanin, eluted before less polar decarboxylated and dehydrogenated derivatives (mostly degradation products of betanin). In IP-HSCCC, the dehydrogenated derivatives were eluted faster then their non-dehydrogenated analogues. It was observed for the fi rst time that betanin and neobetanin (14,15-dehydrogenated betanin), which are present in many plants containing betalains, had reversed elution orders during chromatographic separation by these two techniques

1,2-Hydrogenation and Transhydrogenation Catalyzed by 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans—Kinetics, Isotope Labelling and QM:MM Modelling Studies

Bacteria and fungi that are able to metabolize steroids express 3-ketosteroid-Δ1-dehydrogenases (KstDs). KstDs such as AcmB form Sterolibacterium denitrificans Chol-1 catalyze the enantioselective 1α,2β-dehydrogenation of steroids to their desaturated analogues, e.g., the formation of 1,4-androstadiene-3,17-dione (ADD) from 4-androsten-3,17-dione (AD). The reaction catalyzed by KstD can be reversed if the appropriate electron donor, such as benzyl viologen radical cation, is present. Furthermore, KstDs can also catalyze transhydrogenation, which is the transfer of H atoms between 3-ketosteroids and 1-dehydrosteroids. In this paper, we showed that AcmB exhibits lower pH optima for hydrogenation and dehydrogenation by 3.5–4 pH units than those observed for KstD from Nocardia corallina. We confirmed the enantiospecificity of 1α,2β-hydrogenation and 1α,2β-transhydrogenation catalyzed by AcmB and showed that, under acidic pH conditions, deuterons are introduced not only at 2β but also at the 1α position. We observed a higher degree of H/D exchange at Y363, which activates the C2-H bond, compared to that at FAD, which is responsible for redox at the C1 position. Furthermore, for the first time, we observed the introduction of the third deuteron into the steroid core. This effect was explained through a combination of LC-MS experiments and QM:MM modelling, and we attribute it to a decrease in the enantioselectivity of C2-H activation upon the deuteration of the 2β position. The increase in the activation barrier resulting from isotopic substitution increases the chance of the formation of d3-substituted 3-ketosteroids. Finally, we demonstrate a method for the synthesis of 3-ketosteroids chirally deuterated at 1α,2β positions, obtaining 1α,2β-d2-4-androsten-3,17-dione with a 51% yield (8.61 mg)

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

Background: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (− 8.40 kcal/mol) or diosgenone (− 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 μM) than to AD (KmA = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M−1 s−1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M−1 s−1). Conclusions: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed

Structural and spectroscopic characterisation of bis-ligand complexes of iron(II), nickel(II) and nickel(III) with the hydrotris(methimazolyl)borate anion: soft S-6 donor sets generating a weak ligand field

Reaction of iron(II) and nickel(II) halides with sodium hydrotris(methimazolyl) borate (NaTm) results in the formation of 2 : 1 complexes [M(Tm)(2)]. Crystallographic characterisation reveals in both cases trigonally distorted octahedral geometry in an S-6 donor set. Spectroscopic properties indicate that the Tm ligand generates a weak ligand field, with Dq between that of H2O and Cl- and this is confirmed by the high-spin configuration of the iron(II) complex. Both complexes display classical paramagnetic behaviour. Mossbauer spectroscopy of the iron complex is also consistent with a high-spin complex. Reaction of NiCl2.6H(2)O with [Tl(Tm)] results in a small quantity of a material which is crystallographically demonstrated to be [Ni(Tm)(2)]Br