Mutase Activity Research Articles

New-to-nature CO2-dependent acetyl-CoA assimilation enabled by an engineered B12-dependent acyl-CoA mutase

Acetyl-CoA is a key metabolic intermediate and the product of various natural and synthetic one-carbon (C1) assimilation pathways. While an efficient conversion of acetyl-CoA into other central metabolites, such as pyruvate, is imperative for high biomass yields, available aerobic pathways typically release previously fixed carbon in the form of CO2. To overcome this loss of carbon, we develop a new-to-nature pathway, the Lcm module, in this study. The Lcm module provides a direct link between acetyl-CoA and pyruvate, is shorter than any other oxygen-tolerant route and notably fixes CO2, instead of releasing it. The Lcm module relies on the new-to-nature activity of a coenzyme B12-dependent mutase for the conversion of 3-hydroxypropionyl-CoA into lactyl-CoA. We demonstrate Lcm activity of the scaffold enzyme 2-hydroxyisobutyryl-CoA mutase from Bacillus massiliosenegalensis, and further improve catalytic efficiency 10-fold by combining in vivo targeted hypermutation and adaptive evolution in an engineered Escherichia coli selection strain. Finally, in a proof-of-principle, we demonstrate the complete Lcm module in vitro. Overall, our work demonstrates a synthetic CO2-incorporating acetyl-CoA assimilation route that expands the metabolic solution space of central carbon metabolism, providing options for synthetic biology and metabolic engineering.

A New Tailored Nanodroplet Carrier of Astaxanthin Can Improve Its Pharmacokinetic Profile and Antioxidant and Anti-Inflammatory Efficacies.

Astaxanthin (ATX) is a carotenoid nutraceutical with poor bioavailability due to its high lipophilicity. We tested a new tailored nanodroplet capable of solubilizing ATX in an oil-in-water micro-environment (LDS-ATX) for its capacity to improve the ATX pharmacokinetic profile and therapeutic efficacy. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the pharmacokinetics of ATX and LDS-ATX, superoxide mutase (SOD) activity to determine their antioxidant capacity, protein carbonylation and lipid peroxidation to compare their basal and lipopolysaccharide (LPS)-induced oxidative damage, and ELISA-based detection of IL-2 and IFN-γ to determine their anti-inflammatory capacity. ATX and LDS-ATX corrected only LPS-induced SOD inhibition and oxidative damage. SOD activity was restored only by LDS-ATX in the liver and brain and by both ATX and LDS-ATX in muscle. While in the liver and muscle, LDS-ATX attenuated oxidative damage to proteins and lipids better than ATX; only oxidative damage to lipids was preferably corrected by LDS-ATX in the brain. IL-2 and IFN-γ pro-inflammatory response was corrected by LDS-ATX and not ATX in the liver and brain, but in muscle, the IL-2 response was not corrected and the IFN-γ response was mitigated by both. These results strongly suggest an organ-dependent improvement of ATX bioavailability and efficacy by the LDS-ATX nanoformulation.

Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

Functional vitamin B12 deficiency is a consistent feature in hospital admissions for neurological disorders due to the use of nitrous oxide

Introduction: Misuse of inhaled nitrous oxide is a growing concern in France. It is known to alter concentrations of vitamin B12, which is required as a cofactor for methionine synthase and methylmalonyl-CoA mutase activity. Hence, measurement of the concentrations of cobalamin metabolism biomarkers, including vitamin B12, homocysteine, and methylmalonic acid, could assist in the management of patients with a complex clinical presentation or in those who deny the consumption of nitrous oxide. Methods: We retrospectively collected clinical and biological data in patients hospitalized for nitrous oxide use in a university hospital in southern France between January 2020 and October 2022. Results: Thirty-one patients were identified during 34 months; 79% were men with a median age of 23.7 years. Most (97%) presented with peripheral polyneuropathy and/or myelopathy. The median vitamin B12 concentration was 134.6 pmol/L, with 17 of 31 patients having values less than 145 pmol/L (the lower limit of the normal range). The median plasma folate concentration was 20.1 nmol/L, which is within the normal range. The median plasma homocysteine concentration was 87.7 µmol/L (normal range >15 µmol/L), and the median plasma methylmalonic acid concentration was 3.8 µmol/L (normal range >0.5 µmol/L). Conclusion: Nitrous oxide use is an emerging public health problem in France, as shown by the number of patients admitted to our hospital. The presence of a functional vitamin B12 deficiency was a consistent feature that could be helpful in diagnosis in complex cases.

Novel exported fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: Shikimate pathway enzymes teamed up in no man's land

Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas the order is inverted in β-Proteobacteria. The CM domains share 15% to 20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (∗MtCM), and the CDT domains 40% to 60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). Invitro kinetics revealed a Km <7μM, much lower than for ∗MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway's end product Phe, and no catalytic benefit of the domain fusion compared with engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4Å resolution, respectively. Neither the crystal structures nor the size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for the cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.

Enzymatic reconstitution of salicylate formation in promysalin biosynthesis

Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future.

Erythrocyte ENT1-AMPD3 Axis is an Essential Purinergic Hypoxia Sensor and Energy Regulator Combating CKD in a Mouse Model.

Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. In this study, we demonstrated in a mouse model that erythrocyte ENT1-AMPD3 is a master energy regulator of the intracellular purinergic hypoxic compensatory response that promotes rapid energy supply from extracellular adenosine, eAMPK-dependent metabolic reprogramming, and O 2 delivery, which combat renal hypoxia and progression of CKD. ENT1-AMPD3-AMPK-BPGM comprise a group of circulating erythroid-specific biomarkers, providing early diagnostic and novel therapeutic targets for CKD. Hypoxia drives kidney damage and progression of CKD. Although erythrocytes respond rapidly to hypoxia, their role and the specific molecules sensing and responding to hypoxia in CKD remain unclear. Mice with an erythrocyte-specific deficiency in equilibrative nucleoside transporter 1 ( eEnt1-/- ) and a global deficiency in AMP deaminase 3 ( Ampd3-/- ) were generated to define their function in two independent CKD models, including angiotensin II (Ang II) infusion and unilateral ureteral obstruction (UUO). Unbiased metabolomics, isotopic adenosine flux, and various biochemical and cell culture analyses coupled with genetic studies were performed. Translational studies in patients with CKD and cultured human erythrocytes examined the role of ENT1 and AMPD3 in erythrocyte function and metabolism. eEnt1-/- mice display severe renal hypoxia, kidney damage, and fibrosis in both CKD models. The loss of eENT1-mediated adenosine uptake reduces intracellular AMP and thus abolishes the activation of AMPK α and bisphosphoglycerate mutase (BPGM). This results in reduced 2,3-bisphosphoglycerate and glutathione, leading to overwhelming oxidative stress in eEnt1-/- mice. Excess reactive oxygen species (ROS) activates AMPD3, resulting in metabolic reprogramming and reduced O 2 delivery, leading to severe renal hypoxia in eEnt1-/- mice. By contrast, genetic ablation of AMPD3 preserves the erythrocyte adenine nucleotide pool, inducing AMPK-BPGM activation, O 2 delivery, and antioxidative stress capacity, which protect against Ang II-induced renal hypoxia, damage, and CKD progression. Translational studies recapitulated the findings in mice. eENT1-AMPD3, two highly enriched erythrocyte purinergic components that sense hypoxia, promote eAMPK-BPGM-dependent metabolic reprogramming, O 2 delivery, energy supply, and antioxidative stress capacity, which mitigates renal hypoxia and CKD progression.

Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding

Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear. Here we report the first crystal structures of GT75 family NDP-pyranose mutases. The novel structures of GT75 member MtdL in complex with Mn2+ and GDP, GDP-D-glucopyranose, GDP-L-fucopyranose, GDP-L-fucofuranose, respectively, combined with site-directed mutagenesis studies, reveal key residues involved in Mn2+ coordination, substrate binding, and catalytic reactions. We also provide a possible catalytic mechanism for this unique type of NDP-pyranose mutase. Taken together, our results highlight key elements of an enzyme family important for furanose biosynthesis.

Biochemical Impact Inflicted by Aphid Induced Urdbean Leaf Crinkle Disease (ULCD) Infection on Different Blackgram Genotypes

Aims: To investigate the variations in total protein content and anti-oxidative enzymes in blackgram genotypes showing various levels of resistance against aphid-mediated Urdbean leaf crinkle disease (ULCD).&#x0D; Study Design: Completely Randomized Design.&#x0D; Place and Duration of Study: The studies were carried out at the Insectary belonging to the Department of Agricultural Entomology and the Centre of Innovation at Agricultural College and Research Institute, Madurai, Tamil Nadu, India in January 2021.&#x0D; Methodology: The research was conducted on seven blackgram genotypes exhibiting different levels of resistance to ULCD. A pot culture study was established and the test plants were subjected for inoculation of urdbean leaf crinkle virus (ULCV) via feeding by 10 viruliferous Aphis craccivora adults/plant. The alterations in the total protein content, superdioxide mutase (SOD), catalase (CAT) and peroxidase (PO) activity was estimated using spectrophotometric methods in comparison to healthy genotypes on 0, 15 and 30 days after inoculation (DAI).&#x0D; Results: No discernible increase in the levels of protein content was seen in either of ULCV inoculated or un-inoculated genotypes on the day of the inoculation. At post inoculation, leaf protein content of ULCV highly susceptible genotypes (VBN 8 and T9) was only marginally higher than that of resistant genotypes (CO 5 and CO 6), whereas in moderately resistant genotype (APK 1) it had only slightly increased. The SOD activity showed only a non-significant drop between inoculated versus un-inoculated plant leaves in highly susceptible VBN 8 and T9 genotypes. At 15 and 30 days post inoculation, a significantly less SOD activity was observed, but PO inversely increased compared to the latter under ULCV inoculated versus un-inoculated conditions in ULCV resistant CO 5 and CO 6 genotypes. While the susceptible ADT 5 and ADT 6 and highly susceptible VBN 8 and T9 genotypes did not show any significant increase in PO levels between inoculated versus un-inoculated conditions. Interestingly, ULCV inoculation considerably decreased the reduced CAT activity both in resistant and susceptible genotypes, when compared to the un-inoculated healthy ones.&#x0D; Conclusion: Fluctuations in the levels of antioxidant enzymes as well as the total protein content was significant only at 15 Days post inoculation. The ULCV infection tends to increase the total protein content in inoculated plants compared to un-inoculated plants. The aphid transmission of ULCV infection in different blackgram genotypes inflicted considerable increase in PO while decrease in SOD and CAT activities in resistant CO 5 and CO 6 genotypes.

Molecular insight into 2-phosphoglycolate activation of the phosphatase activity of bisphosphoglycerate mutase.

Bisphosphoglycerate mutase (BPGM) is an erythrocyte-specific multifunctional enzyme that is responsible for the regulation of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells through its synthase and phosphatase activities; the latter enzymatic function is stimulated by the endogenous activator 2-phosphoglycolate (2-PG). 2,3-BPG is a natural allosteric effector of hemoglobin (Hb) that is responsible for decreasing the affinity of Hb for oxygen to facilitate tissue oxygenation. Here, crystal structures of BPGM with 2-PG in the presence and absence of 3-phosphoglycerate are reported at 2.25 and 2.48 Å resolution, respectively. Structure analysis revealed a new binding site for 2-PG at the dimer interface for the first time, in addition to the expected active-site binding. Also, conformational non-equivalence of the two active sites was observed as one of the sites was found in an open conformation, with the residues at the active-site entrance, including Arg100, Arg116 and Arg117, and the C-terminus disordered. The kinetic result is consistent with the binding of 2-PG to an allosteric or noncatalytic site as well as the active site. This study paves the way for the rational targeting of BPGM for therapeutic purposes, especially for the treatment of sickle cell disease.

Short-term warming inhibits polyphenol oxidase activity and influences free amino acid accumulation and de novo synthesis of tyrosine in fresh-cut potato

Potatoes cut immediately after being taken out from cold storage easily undergo browning. Researchers have reported that long-term warming alleviates the browning of fresh-cut potatoes. However, long-term warming is not commercially applicable. The present study investigated the effect of short-term warming (SW) at 25 °C for 12 h on the browning of fresh-cut potatoes. The browning index of potatoes stored at 4 °C for 8 months was tested. The analysis revealed that SW efficiently blocked enzymatic browning by inhibiting polyphenol oxidase (PPO) activity, decreasing total phenolic content and malondialdehyde (MDA) content, and increasing peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activity, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azino-bis (3-ethylben zothiazoline) 6-sulphonic acid (ABTS) inhibition rates. Moreover, SW treatment induced the expression of the serine protease inhibitor (SPI) gene and enhanced SPI activity, increased the soluble protein content and decreased free tyrosine, and lysine content in fresh-cut potatoes during the 5 d storage at 2∼4 °C. The contents of glutamic acid, aspartic acid, and proline in the fresh-cut potatoes increased after SW treatment. Furthermore, the amino acid complementation experiments showed that tyrosine and lysine aggravated browning while glutamic acid and aspartic acid reduced browning. The SW treatment reduced the activity of chorismate mutase (CM), repressed the transcript levels of CM synthesis-related genes (CM1 and CM2), and inhibited free tyrosine synthesis. These results indicate that SW treatment alleviates the browning of potato tubers after cold storage probably due to its influence on PPO, metabolism of tyrosine, other FAAs and phenolics, and antioxidants.

Inherited defects of cobalamin metabolism.

Cobalamin (vitamin B12) is required for activity of the enzymes methylmalonyl-CoA mutase and methionine synthase in human cells. Inborn errors affecting cobalamin uptake or metabolism are characterized by accumulation of the substrates for these enzymes, methylmalonic acid and homocysteine, in blood and urine. Inborn errors affecting synthesis of the adenosylcobalamin coenzyme required by methylmalonyl-CoA mutase (cblA and cblB) result in isolated methylmalonic aciduria; inborn errors affecting synthesis of the methylcobalamin coenzyme required by methionine synthase (cblE and cblG) result in isolated homocystinuria. Combined methylmalonic aciduria and homocystinuria is seen in patients with impaired intestinal cobalamin absorption (intrinsic factor deficiency, Imerslund-Gräsbeck syndrome) and with defects affecting synthesis of both cobalamin coenzymes (cblC, cblD, cblF and cblJ). A series of disorders caused by pathogenic variant mutations affecting gene regulators (transcription factors) of the MMACHC gene have recently been described (HCFC1 [cblX disorder] and deficiencies of THAP11, and ZNF143 [the cblK disorder]).

Anthocyanin extract from Lycium ruthenicum enhanced production of biomass and polysaccharides during submerged fermentation of Agaricus bitorquis (Qu\xe9l.) Sacc. Chaidam

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC) is a wild edible fungus uniquely found in the Tibet Plateau. ABSC is rich in polysaccharides that are considered biologically active. This study aimed to determine the feasibility of enhancing exopolysaccharide (EPS) production by ABSC in shake flask culture by supplementing the fermentation medium with anthocyanin extract. Different concentrations of Lycium ruthenicum Murr. (LRM) anthocyanin crude extract were tested on ABSC fermentation. The activity of phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM), and phosphomannose isomerase (PMI), enzymes presumably involved in EPS synthesis by ABSC, was determined. ABSC transcriptomic profile in response to the presence of anthocyanins during fermentation was also investigated. LRM anthocyanin crude extract (0.06 mg/mL) was most effective in increasing EPS content and mycelial biomass (by 208.10% and 105.30%, respectively, P < 0.01). The activity of PGI, PGM, and PMI was increased in a medium where LRM anthocyanin extract and its main components (proanthocyanidins and petunia anthocyanin) were added. RNA-Seq analysis showed that 349 genes of ABSC were differentially expressed during fermentation in the medium containing anthocyanin extract of LRM; 93 genes were up-regulated and 256 genes down-regulated. From gene ontology enrichment analysis, differentially expressed genes were mostly assigned to carbohydrate metabolism and signal transduction categories. Collectively, LRM anthocyanins extract positively affected EPS production and mycelial biomass during ABSC fermentation. Our study provides a novel strategy for improving EPS production and mycelial growth during ABSC liquid submerged fermentation.

MUT gene variants in patients with methylmalonic acidemia in Bangladeshi population and their distinguishing metabolic profiles

Methylmalonic acidemia (MMA) is a rare inborn error of organic acid metabolism presented with wide range of clinical features from mild to severe life-threatening conditions. It is caused mostly due to defective activity of the enzyme methylmalonyl-CoA mutase (MCM), which is encoded by the MUT gene. In this study we analyzed the clinical and biochemical features as well as mutation spectrum in the coding regions (exon 2–13) of the MUT gene and their adjacent intronic consensus splice sites in unrelated MMA patients and healthy individuals. We identified 14 mutations in the MUT gene among which two (c.856G > C and c.1676 + 15C > T) were not reported earlier. Bioinformatics tools were used to explore the molecular consequences of these 14 mutations on MCM activity and correlated these predictions to the phenotypic severities of the patients. Our analysis suggest that a novel mutation c.856G > C (p.E286Q) and a previously reported mutation c.1837C > T (R613C) may have disease causing effect and play important role in methylmalonic acidemia. In addition, we compared the profiles of 79 metabolic features (47 individual metabolite concentrations and 32 ratios) between the MMA patients and healthy controls. Although elevated levels of propionylcarnitine (C3) and ratio of propionylcarnitine (C3) to acetylcarnitine (C2) in blood are considered as the diagnostic features of MMA, this study could clearly distinguish between the MMA patients and the controls based on C3 levels only, but not C3/C2 in a statistically significant manner. In addition to C3, the ratio of argininosuccinic acid (ASA), argininosuccinic acid/arginine (ASA/Arg), and 3-hydroxyisovaleryl−/2-methyl-3-hydroxybutyryl-carnitine/propionylcarnitine (C5OH/C3) were clearly distinguishable between the groups with ≥2 fold changes in concentration.

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte–specific ADORA2B (eAdora2b−/−) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b−/− mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B–BPGM axis is a key component for anti-aging and anti-age–related functional decline.

Structural analysis of EhPSP in complex with 3-phosphoglyceric acid from Entamoeba histolytica reveals a basis for its lack of phosphoglycerate mutase activity

Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.

Plasma 3-hydroxyisobutyrate (3-HIB) and methylmalonic acid (MMA) are markers of hepatic mitochondrial fatty acid oxidation in male Wistar rats

ObjectiveDiscovery of specific markers that reflect altered hepatic fatty acid oxidation could help to detect an individual's risk of fatty liver, type 2 diabetes and cardiovascular disease at an early stage. Lipid and protein metabolism are intimately linked, but our understanding of this crosstalk remains limited. MethodsIn male Wistar rats, we used synthetic fatty acid analogues (3-thia fatty acids) as a tool to induce hepatic fatty acid oxidation and mitochondrial biogenesis, to gain new insight into the link between fatty acid oxidation, amino acid metabolism and TCA cycle-related intermediate metabolites in liver and plasma. ResultsRats treated with 3-thia fatty acids had 3-fold higher hepatic, but not adipose and skeletal muscle, expression of the thioesterase 3-hydroxyisobutyryl-CoA hydrolase (Hibch), which controls the formation of 3-hydroxyisobutyrate (3-HIB) in the valine degradation pathway. Consequently, 3-thia fatty acid-stimulated hepatic fatty acid oxidation and ketogenesis was accompanied by decreased plasma 3-HIB and increased methylmalonic acid (MMA) concentrations further downstream in BCAA catabolism. The higher plasma MMA corresponded to higher MMA-CoA hydrolase activity and hepatic expression of GTP-specific succinyl-CoA synthase (Suclg2) and succinate dehydrogenase (Sdhb), and lower MMA-CoA mutase activity. Plasma 3-HIB correlated positively to plasma and hepatic concentrations of TAG, plasma total fatty acids, plasma NEFA and insulin/glucose ratio, while the reverse correlations were seen for MMA. ConclusionOur study provides new insight into TCA cycle-related metabolic changes associated with altered hepatic fatty acid flux, and identifies 3-HIB and MMA as novel circulating markers reflective of mitochondrial β-oxidation in male Wistar rats.

Kinetic Studies of a Coenzyme B12 Dependent Reaction Catalyzed by Glutamate Mutase from &amp;lt;i&amp;gt;Clostridium cochlearium&amp;lt;/i&amp;gt;

The coenzyme B12 dependent glutamate mutase is composed of two apoenzyme proteins subunits; S and E2, which while either fused or separate assemble with coenzyme B12 to form an active holoenzyme (E2S2-B12) for catalyzing the reversible isomerization between (S)-glutamate and (2S, 3S)-3-methylas- partate. In order to assay the activity of glutamate mutase by UV spectrophotometry, this reaction is often coupled with methylaspartase which deaminates (2S, 3S)-3-methylaspartate to form mesaconate (λmax = 240 nm, ?240 = 3.8 mM?1?cm?1). The activities of different reconstitutions of glutamate mutase from separate apoenzyme components S and E in varied amounts of coenzyme B12 and adenosylpeptide B12 as cofactors were measured by this assay and used to reveal the binding properties of the cofactor by the Michaelis- Menten Method. The values of Km for coenzyme B12 in due to reconstitutions of holoenzyme in 2, 7 and 14 S: E were determined as; 1.12 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B12; 1.07 ± 0.04 μM and 0.35 ± 0.05 μM as obtained from respective 2 and 14 S: E compositions of holoenzyme. Analysis of these kinetics results curiously associates the increasing affinity of cofactors to apoenzyme with increased amount of component S used in reconstituting holoenzyme from separate apoenzyme components and cofactor. Moreover, in these studies a new method for assaying the activity of glutamate mutase was developed, whereby glutamate mutase activity is measured via depletion of NADH (λmax = 340 nm, ?340 = 6.3 mM?1?cm?1) as determined by UV spectrophotometry after addition of (2S, 3S)-3-methylaspartate and pyruvate to a mixture of E2S2-B12 and two auxiliary holoenzymes system; pyridoxal-5-phosphate dependent glutamate-pyruvate aminotransferase and NADH dependent (R)-2-hydroxyglutarate dehydrogenase. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (S)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (R)-2-hydroxylglutarate dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2S, 3S)-3-methylaspartate in the reaction of glutamate mutase as Km= 7 ± 0.07 mM and kcat= 0.54 ± 0.6 s?1. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, Keq = [(S<span style="font-family:Ve

A New Approach for Second-tier Analysis of Methylmalonic Acid in Dried Blood Spots using Liquid Chromatography Tandem Mass Spectrometry

Introduction: Inactivity or diminished activity of an enzyme Methylmalonyl-CoA mutase (a Cobalamin dependent) enzyme causes inborn error of metabolism named Methylmalonic Acidemia/Aciduria (MMA). Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) based method for the diagnosis of MMA in Newborn Screening (NBS), is often challenging due to the nonspecificity of propionylcarnitine (C3), a primary marker in routine NBS. Aim: To develop a Flow Injection Analysis (FIA) method for the second-tier estimation of Methylmalonic acid in the Dried Blood Spots (DBS) of primary NBS. Materials and Methods: A retrospective NBS study was conducted for a period of two years i.e. (November 2015 to November 2017) at the Paediatrics Research and Genetic Lab of Maulana Azad Medical College associated Lok Nayak Hospital, a Tertiary Care Centre in New Delhi, India. DBS samples were collected by heel- prick method and a second tier detection, quantification of methylmalonic acid was performed by LC-MS/MS on all samples with abnormal C3 levels in primary NBS. Multiple Reaction Monitoring (MRM) mode at m/z 117→73 for MMA and m/z 120→75 for MMA(IS) and isotopic dilution approach was followed for quantification. Results: Intra-assay and inter-assay precision and accuracy was determined at two different levels of MMA (LQC≅2.0 μmol/L and HQC≅10.0 μmol/L), respectively. The Coefficient of Variation (%) for intraday precision ranged between 5.27% to 8.9%. Similarly, for interday it ranged from 4.99% to 9.93%. The average accuracy (%) also falls within (105.4% and 106.1%) for interday and (105.9% and 106.7%) for intraday assay. Stability for samples during storage at different temperature i.e., (fresh, 2-8°C and -20°C) showed long term stability at -20°C storage. The assay was linear over a calibration range of (0.5 to 20.00 μmol/L). Conclusion: The outcome of the present data offers the confidence and reliability in the possible utility of this method for the definitive diagnosis and follows-up of MMA patients.

Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC

BackgroundPHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon–neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW).ResultsWe have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic.ConclusionThe amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.