Mitochondrial Fragmentation and Dysfunction in Type IIx/IIb Diaphragm Muscle Fibers in 24-Month Old Fischer 344 Rats

Sarcopenia is characterized by muscle fiber atrophy and weakness, which may be associated with mitochondrial fragmentation and dysfunction. Mitochondrial remodeling and biogenesis in muscle fibers occurs in response to exercise and increased muscle activity. However, the adaptability mitochondria may decrease with age. The diaphragm muscle (DIAm) sustains breathing, via recruitment of fatigue-resistant type I and IIa fibers. More fatigable, type IIx/IIb DIAm fibers are infrequently recruited during airway protective and expulsive behaviors. DIAm sarcopenia is restricted to the atrophy of type IIx/IIb fibers, which impairs higher force airway protective and expulsive behaviors. The aerobic capacity to generate ATP within muscle fibers depends on the volume and intrinsic respiratory capacity of mitochondria. In the present study, mitochondria in type-identified DIAm fibers were labeled using MitoTracker Green and imaged in 3-D using confocal microscopy. Mitochondrial volume density was higher in type I and IIa DIAm fibers compared with type IIx/IIb fibers. Mitochondrial volume density did not change with age in type I and IIa fibers but was reduced in type IIx/IIb fibers in 24-month rats. Furthermore, mitochondria were more fragmented in type IIx/IIb compared with type I and IIa fibers, and worsened in 24-month rats. The maximum respiratory capacity of mitochondria in DIAm fibers was determined using a quantitative histochemical technique to measure the maximum velocity of the succinate dehydrogenase reaction (SDHmax). SDHmax per fiber volume was higher in type I and IIa DIAm fibers and did not change with age. In contrast, SDHmax per fiber volume decreased with age in type IIx/IIb DIAm fibers. There were two distinct clusters for SDHmax per fiber volume and mitochondrial volume density, one comprising type I and IIa fibers and the second comprising type IIx/IIb fibers. The separation of these clusters increased with aging. There was also a clear relation between SDHmax per mitochondrial volume and the extent of mitochondrial fragmentation. The results show that DIAm sarcopenia is restricted to type IIx/IIb DIAm fibers and related to reduced mitochondrial volume, mitochondrial fragmentation and reduced SDHmax per fiber volume.

(S)-3-Hydroxybutyryl-CoA Dehydrogenase From the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Nitrosopumilus maritimus

Ammonia-oxidizing archaea of the phylum Thaumarchaeota are among the most abundant organisms that exert primary control of oceanic and soil nitrification and are responsible for a large part of dark ocean primary production. They assimilate inorganic carbon via an energetically efficient version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. In this cycle, acetyl-CoA is carboxylated to succinyl-CoA, which is then converted to two acetyl-CoA molecules with 4-hydroxybutyrate as the key intermediate. This conversion includes the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Here, we heterologously produced the protein Nmar_1028 catalyzing this reaction in thaumarchaeon Nitrosopumilus maritimus, characterized it biochemically and performed its phylogenetic analysis. This NAD-dependent dehydrogenase is highly active with its substrate, (S)-3-hydroxybutyryl-CoA, and its low Km value suggests that the protein is adapted to the functioning in the 3-hydroxypropionate/4-hydroxybutyrate cycle. Nmar_1028 is homologous to the dehydrogenase domain of crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase that is present in many Archaea. Apparently, the loss of the dehydratase domain of the fusion protein in the course of evolution was accompanied by lateral gene transfer of 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase from Bacteria. Although (S)-3-hydroxybutyryl-CoA dehydrogenase studied here is neither unique nor characteristic for the HP/HB cycle, Nmar_1028 appears to be the only (S)-3-hydroxybutyryl-CoA dehydrogenase in N. maritimus and is thus essential for the functioning of the 3-hydroxypropionate/4-hydroxybutyrate cycle and for the biology of this important marine archaeon.

Oxidation or dehydrogenation of alpha-hydroxy acids in bioenergetic metabolism: A murburn perspective

Glycolate, lactate, malate, hydroxyglutarate and isocitrate are key alpha-hydroxyacyl metabolic intermediates found in the tissues/cells/organelles of diverse life forms. They are respectively oxidized to glyoxylate, pyruvate, oxaloacetate, ketoglutarate and oxalosuccinate in cell bioenergetic metabolism. These molecules form key junction points for divergent pathways of two to six carbon-backboned molecules (of various classes of biomolecules like carbohydrates, amino acids, etc.). The oxido-reduction of the alpha-hydroxyacyl species is traditionally believed to be carried out by reversible (de)hydrogenases, employing nicotinamide cofactors. Herein, I propose that while the reductive pathway can be mediated in a facile manner by the (de)hydrogenases, the oxidative reaction could more efficiently be coupled with murzyme activities, which employ diffusible reactive (oxygen) species (DRS/DROS/ROS). Such a murburn strategy would enable the system to tide over the highly unfavorable energy barriers of the sequential dehydrogenase reaction (~450 kJ/mol, or more!), to give kinetically viable bimolecular reactions catering to cellular needs. Further, such a scheme does not necessitate any ‘intelligent governance’ or ‘smart decision-making’ of/by the pertinent redox enzymes.

Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2

ABSTRACT Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut– phenotype. The Mut– phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut– strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut– increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

Rid enhances the 6-hydroxypseudooxynicotine dehydrogenase reaction in nicotine degradation by Agrobacterium tumefaciens S33

Agrobacterium tumefaciens S33 degrades nicotine through a hybrid of the pyridine and pyrrolidine pathways. The oxidation of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxypseudooxynicotine dehydrogenase (Pno) is an important step in the breakdown of the N-heterocycle in this pathway. Although Pno has been characterized, the reaction is not fully understood, i.e., it starts at a high speed, followed by a rapid drop in the reaction rate, leading to the formation of a very small amount of product. In this study, we speculated that an unstable imine intermediate is produced in the reaction, which may be toxic to the metabolism. We found that a Rid protein (designated as Rid-NC) encoded by a gene in the nicotine-degrading gene cluster enhanced the reaction. Rid is a widely distributed family of small proteins with various functions, and some subfamilies have deaminase activity to eliminate the toxicity of the reactive intermediate imine. Biochemical analyses showed that Rid-NC relieved the toxicity of the presumable imine intermediate produced in the Pno reaction, and that, in the presence of Rid-NC, Pno maintained a high activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene caused a slower growth of strain S33 on nicotine. The mechanism of Rid-NC-mediated detoxification of the imine intermediate was discussed. A phylogenetic analysis indicated that Rid-NC belongs to the rarely studied Rid6 subfamily. These results further our understanding of the biochemical mechanism of nicotine degradation and provide new insights into the function of the Rid6 subfamily proteins. IMPORTANCE Rid is a family of proteins that participate in metabolite-damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. Rid-NC hydrolyzed the presumable reactive imine intermediate produced in the reaction to remove its toxicity on Pno. The finding furthers our understanding of the metabolic process of the toxic N-heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of N-heterocyclic aromatic alkaloids, in addition to the amino acid metabolism, and that Rid6-subfamily proteins also have deaminase activity, similar to RidA subfamily. The ability of reactive imines to damage a non-pyridoxal-5′-phosphate-dependent enzyme was reported. This study provides new insights into the function of the Rid family of proteins.

ВПЛИВ ЗАМІЩЕННОГО НІКОТИНАМІДУ ТА ЙОГО МОЖЛИВИХ МЕТАБОЛІТІВ НА АКТИВНІСТЬ ФЕРМЕНТІВ ОБМІНУ ЕТАНОЛУ

To study the influence of N-phenyl-N-(1-cyclopropylethyl)nicotinamide and its possible metabolites: hydrochlorides of N-(1-cyclopropylethyl)amine and N-phenyl-N-(1-cyclopropylethyl)amine - on the activity of main ethanol oxidation enzymes in vitro and kinetic nature of their interaction. The studies were carried out using alcohol dehydrogenase and aldehyde dehydrogenase of rat liver subcellular fractions, which were obtained by differential centrifugation. The enzyme activity was determined spectrophotometrically. The kinetic nature of alcohol dehydrogenase and isozyme form of aldehyde dehydrogenase interaction with substituted nicotinamide was investigated in the concentration range of 25-100 μM. The research results were processed by the Lineweaver-Burk method. Studies have shown that N-phenyl-N-(1-cyclopropylethyl)nicotinamide is able to reduce the rate of the reverse alcohol dehydrogenase reaction of acetaldehyde reduction to ethanol in the presence of NADH by 46% with an inhibition constant 53 μM. The activity of soluble mitochondrial aldehyde dehydrogenase was suppressed by 50% with an inhibition constant 108 μM. The kinetic nature of the substituted nicotinamide interaction with enzymes at saturating concentrations of the reaction cofactors NADH and NAD+ is quite complex. Allosteric effects can play a significant role in enzymatic activity. Possible metabolites of the compound - hydrochlorides of N-(1-cyclopropylethyl)- and N-phenyl-N-(1-cyclopropylethyl)amine – didn`t significantly influence on ethanol metabolism enzymes activity. A new inhibitor of the rate of the reverse alcohol dehydrogenase reaction and the activity of soluble mitochondrial isozyme form of aldehyde dehydrogenase, which lead to the accumulation of acetaldehyde in the body, has been discovered. N-phenyl-N-(1-cyclopropylethyl)nicotinamide can be used as a potential antialcohol sensitizing drug after research in vivo.

Structure: Function Studies of the Cytosolic, Mo- and NAD+-Dependent Formate Dehydrogenase from Cupriavidus necator

Here, we report recent progress our laboratories have made in understanding the maturation and reaction mechanism of the cytosolic and NAD+-dependent formate dehydrogenase from Cupriavidus necator. Our recent work has established that the enzyme is fully capable of catalyzing the reverse of the physiological reaction, namely, the reduction of CO2 to formate using NADH as a source of reducing equivalents. The steady-state kinetic parameters in the forward and reverse directions are consistent with the expected Haldane relationship. The addition of an NADH-regenerating system consisting of glucose and glucose dehydrogenase increases the yield of formate approximately 10-fold. This work points to possible ways of optimizing the reverse of the enzyme’s physiological reaction with commercial potential as an effective means of CO2 remediation. New insight into the maturation of the enzyme comes from the recently reported structure of the FdhD sulfurase. In E. coli, FdhD transfers a catalytically essential sulfur to the maturing molybdenum cofactor prior to insertion into the apoenzyme of formate dehydrogenase FdhF, which has high sequence similarity to the molybdenum-containing domain of the C. necator FdsA. The FdhD structure suggests that the molybdenum cofactor may first be transferred from the sulfurase to the C-terminal cap domain of apo formate dehydrogenase, rather than being transferred directly to the body of the apoenzyme. Closing of the cap domain over the body of the enzymes delivers the Mo-cofactor into the active site, completing the maturation of formate dehydrogenase. The structural and kinetic characterization of the NADH reduction of the FdsBG subcomplex of the enzyme provides further insights in reversing of the formate dehydrogenase reaction. Most notably, we observe the transient formation of a neutral semiquinone FMNH·, a species that has not been observed previously with holoenzyme. After initial reduction of the FMN of FdsB by NADH to the hydroquinone (with a kred of 680 s−1 and Kd of 190 µM), one electron is rapidly transferred to the Fe2S2 cluster of FdsG, leaving FMNH·. The Fe4S4 cluster of FdsB does not become reduced in the process. These results provide insight into the function not only of the C. necator formate dehydrogenase but also of other members of the NADH dehydrogenase superfamily of enzymes to which it belongs.