RNAase III-Type Enzyme Dicer Regulates Mitochondrial Fatty Acid Oxidative Metabolism in Cardiac Mesenchymal Stem Cells

Cardiac mesenchymal stem cells (C-MSC) play a key role in maintaining normal cardiac function under physiological and pathological conditions. Glycolysis and mitochondrial oxidative phosphorylation predominately account for energy production in C-MSC. Dicer, a ribonuclease III endoribonuclease, plays a critical role in the control of microRNA maturation in C-MSC, but its role in regulating C-MSC energy metabolism is largely unknown. In this study, we found that Dicer knockout led to concurrent increase in both cell proliferation and apoptosis in C-MSC compared to Dicer floxed C-MSC. We analyzed mitochondrial oxidative phosphorylation by quantifying cellular oxygen consumption rate (OCR), and glycolysis by quantifying the extracellular acidification rate (ECAR), in C-MSC with/without Dicer gene deletion. Dicer gene deletion significantly reduced mitochondrial oxidative phosphorylation while increasing glycolysis in C-MSC. Additionally, Dicer gene deletion selectively reduced the expression of β-oxidation genes without affecting the expression of genes involved in the tricarboxylic acid (TCA) cycle or electron transport chain (ETC). Finally, Dicer gene deletion reduced the copy number of mitochondrially encoded 1,4-Dihydronicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase core subunit 6 (MT-ND6), a mitochondrial-encoded gene, in C-MSC. In conclusion, Dicer gene deletion induced a metabolic shift from oxidative metabolism to aerobic glycolysis in C-MSC, suggesting that Dicer functions as a metabolic switch in C-MSC, which in turn may regulate proliferation and environmental adaptation

Sulfite: Cytochrome \u3cem\u3ec\u3c/em\u3e Oxidoreductase from \u3cem\u3eThiobacillus novellus\u3c/em\u3e

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC1.8.2.1). Here we show that the enzyme from Thiobacillus novellus is a periplasmically located αβ heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa mono-heme cytochrome c 552 subunit (midpoint redox potential, E m8.0 = +280 mV). The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K m values for sulfite and cytochrome c 550 were determined to be 27 and 4 μm, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorABgenes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream ofsorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any knownc-type cytochromes

Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly

The final version of this article is available at the link below.Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an ‘average’ 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.This work was partly supported by EPSRC (under grants GR/R99393/01 and EP/C015452/1)

Systematic Chemoenzymatic Synthesis of O-Sulfated Sialyl Lewis x Antigens.

O-Sulfated sialyl Lewis x antigens play important roles in nature. However, due to their structural complexity, they are not readily accessible by either chemical or enzymatic synthetic processes. Taking advantage of a bacterial sialyltransferase mutant that can catalyze the transfer of different sialic acid forms from the corresponding sugar nucleotide donors to Lewis x antigens which are fucosylated glycans as well as an efficient one-pot multienzyme (OPME) sialylation system, O-sulfated sialyl Lewis x antigens containing different sialic acid forms and O-sulfation at different locations were systematically synthesized by chemoenzymatic methods

The Urea Carboxylase and Allophanate Hydrolase Activities of Urea Amidolyase Are Functionally Independent

Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO2. UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function

The dynamic organization of fungal acetyl-CoA carboxylase

Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control

Synthesis of N-Glycolylneuraminic Acid (Neu5Gc) and Its Glycosides.

Sialic acids constitute a family of negatively charged structurally diverse monosaccharides that are commonly presented on the termini of glycans in higher animals and some microorganisms. In addition to N-acetylneuraminic acid (Neu5Ac), N-glycolyl neuraminic acid (Neu5Gc) is among the most common sialic acid forms in nature. Nevertheless, unlike most animals, human cells loss the ability to synthesize Neu5Gc although Neu5Gc-containing glycoconjugates have been found on human cancer cells and in various human tissues due to dietary incorporation of Neu5Gc. Some pathogenic bacteria also produce Neu5Ac and the corresponding glycoconjugates but Neu5Gc-producing bacteria have yet to be found. In addition to Neu5Gc, more than 20 Neu5Gc derivatives have been found in non-human vertebrates. To explore the biological roles of Neu5Gc and its naturally occurring derivatives as well as the corresponding glycans and glycoconjugates, various chemical and enzymatic synthetic methods have been developed to obtain a vast array of glycosides containing Neu5Gc and/or its derivatives. Here we provide an overview on various synthetic methods that have been developed. Among these, the application of highly efficient one-pot multienzyme (OPME) sialylation systems in synthesizing compounds containing Neu5Gc and derivatives has been proven as a powerful strategy

Richard Nelson Perham. 27 April 1937—14 February 2015

Richard Nelson Perham, FRS, FMedSci, FRSA, was a British professor of structural biochemistry. He undertook his academic career at the University of Cambridge, holding positions as lecturer, reader, chair and head of the Department of Biochemistry, as well as becoming Master of St John's College. Perham published close to 300 scientific papers on protein structure and function, with a focus on mechanistic enzymology, particularly how large multienzyme complexes and flavin-containing enzymes work. He is most renowned for determining how reactive intermediates are transferred between enzyme active sites, for alterations of coenzyme and substrate specificity by his pioneering use of protein engineering and for developing protein display methodologies. Married to Nancy Lane-Perham, and with their two children, Perham enjoyed a full and active life in Cambridge and St John's College. He was a keen participator and supporter of sport and enjoyed art, literature, theatre and music. Perham was a vocal and active champion of equal opportunity in education. His legacy to science is a greater understanding of how enzymes work. His legacy to scientists is as a role model of how to attain the highest levels of achievement while maintaining a sense of personal modesty and a keen support for others