Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

This study investigated metabolic responses inSynechocystissp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O2evolution and ATP/NADPH generation by linear electron flow. Based on13C-metabolic flux analysis (13C-MFA) and RNA sequencing, we have found thatSynechocystissp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO2and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.

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Unconventional biochemical regulation of the oxidative pentose phosphate pathway in the model cyanobacterium Synechocystis sp. PCC 6803

Biochemical Journal ◽

10.1042/bcj20200038 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1309-1321

Author(s):

Shoki Ito ◽

Takashi Osanai

Keyword(s):

Tca Cycle ◽

Pentose Phosphate ◽

Succinic Semialdehyde ◽

Synechocystis 6803 ◽

Succinic Semialdehyde Dehydrogenase ◽

Metabolite Production ◽

Semialdehyde Dehydrogenase ◽

Biochemical Regulation ◽

Synechocystis Sp ◽

Pcc 6803

Metabolite production from carbon dioxide using sugar catabolism in cyanobacteria has been in the spotlight recently. Synechocystis sp. PCC 6803 (Synechocystis 6803) is the most studied cyanobacterium for metabolite production. Previous in vivo analyses revealed that the oxidative pentose phosphate (OPP) pathway is at the core of sugar catabolism in Synechocystis 6803. However, the biochemical regulation of the OPP pathway enzymes in Synechocystis 6803 remains unknown. Therefore, we characterized a key enzyme of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PDH), and related enzymes from Synechocystis 6803. Synechocystis 6803 G6PDH was inhibited by citrate in the oxidative tricarboxylic acid (TCA) cycle. Citrate has not been reported as an inhibitor of G6PDH before. Similarly, 6-phosphogluconate dehydrogenase, the other enzyme from Synechocystis 6803 that catalyzes the NADPH-generating reaction in the OPP pathway, was inhibited by citrate. To understand the physiological significance of this inhibition, we characterized succinic semialdehyde dehydrogenase (SSADH) from Synechocystis 6803 (SySSADH), which catalyzes one of the NAD(P)H generating reactions in the oxidative TCA cycle. Similar to isocitrate dehydrogenase from Synechocystis 6803, SySSADH specifically catalyzed the NADPH-generating reaction and was not inhibited by citrate. The activity of SySSADH was lower than that of other bacterial SSADHs. Previous and this studies revealed that unlike the OPP pathway, the oxidative TCA cycle is a pathway with low efficiency in NADPH generation in Synechocystis 6803. It has, thus, been suggested that to avoid NADPH overproduction, the OPP pathway dehydrogenase activity is repressed when the flow of the oxidative TCA cycle increases in Synechocystis 6803.

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Functional γ-Aminobutyrate Shunt in Listeria monocytogenes: Role in Acid Tolerance and Succinate Biosynthesis

Applied and Environmental Microbiology ◽

10.1128/aem.02184-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 74-80 ◽

Cited By ~ 40

Author(s):

Conor Feehily ◽

Conor P. O'Byrne ◽

Kimon Andreas G. Karatzas

Keyword(s):

Listeria Monocytogenes ◽

Crude Protein ◽

Tricarboxylic Acid Cycle ◽

Acid Tolerance ◽

Succinic Semialdehyde ◽

Succinic Semialdehyde Dehydrogenase ◽

Gaba Shunt ◽

Content Type ◽

Semialdehyde Dehydrogenase ◽

Acidic Conditions

ABSTRACTListeria monocytogenes, the causative agent of human listeriosis, is known for its ability to withstand severe environmental stresses. The glutamate decarboxylase (GAD) system is one of the principal systems utilized by the bacterium to cope with acid stress, a reaction that produces γ-aminobutyrate (GABA) from glutamate. Recently, we have shown that GABA can accumulate intracellularly under acidic conditions, even under conditions where no extracellular glutamate-GABA exchange is detectable. The GABA shunt, a pathway that metabolizes GABA to succinate, has been described for several other bacterial genera, and the present study sought to determine whetherL. monocytogeneshas this metabolic capacity, which, if present, could provide a possible route for succinate biosynthesis inL. monocytogenes. Using crude protein extracts fromL. monocytogenesEGD-e, we show that this strain exhibits activity for the two main enzyme reactions in the GABA shunt, GABA aminotransferase (GABA-AT) and succinic semialdehyde dehydrogenase (SSDH). Two genes were identified as candidates for encoding these enzyme activities,argD(GABA-AT) andlmo0913(SSDH). Crude protein extracts prepared from a mutant lacking a functionalargDgene significantly reduced GABA-AT activity, while anlmo0913mutant lost all detectable SSDH activity. The deletion oflmo0913increased the acid tolerance of EGD-e and showed an increased accumulation of intracellular GABA, suggesting that this pathway plays a significant role in the survival of this pathogen under acidic conditions. This is the first report of such a pathway in the genusListeria, which highlights an important link between metabolism and acid tolerance and also presents a possible compensatory pathway to partially overcome the incomplete tricarboxylic acid cycle ofListeria.

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Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase

Applied and Environmental Microbiology ◽

10.1128/aem.03987-14 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5593-5603 ◽

Cited By ~ 11

Author(s):

Sunil Kumar ◽

Tejaswani Saragadam ◽

Narayan S. Punekar

Keyword(s):

Aspergillus Niger ◽

Amine Oxidase ◽

Krebs Cycle ◽

Arginine Decarboxylase ◽

Mass Spectrometry Analysis ◽

Succinic Semialdehyde ◽

Succinic Semialdehyde Dehydrogenase ◽

Content Type ◽

Spectrometry Analysis ◽

Semialdehyde Dehydrogenase

ABSTRACTAgmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds inAspergillus nigerwas explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow onl-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase inA. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grownA. nigermycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization inA. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism.A. nigerGBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from theA. nigerNCIM 565 genome was located and cloned. Subsequent expression of GBase in bothEscherichia coliandA. nigeralong with its disruption inA. nigerfunctionally defined the GBase locus (gbu) in theA. nigergenome.

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Succinic Semialdehyde Dehydrogenase Deficiency: Review of the Natural History Study

Journal of Child Neurology ◽

10.1177/0883073820981262 ◽

2021 ◽

pp. 088307382098126

Author(s):

Phillip L. Pearl ◽

Melissa L. DiBacco ◽

Christos Papadelis ◽

Thomas Opladen ◽

Ellen Hanson ◽

...

Keyword(s):

Natural History ◽

Psychiatric Symptoms ◽

Epileptiform Activity ◽

Laboratory Data ◽

Standard Of Care ◽

Succinic Semialdehyde ◽

Succinic Semialdehyde Dehydrogenase ◽

Natural History Study ◽

Semialdehyde Dehydrogenase ◽

Verbal Score

Objective: The SSADHD Natural History Study was initiated in 2019 to define the natural course and identify biomarkers correlating with severity. Methods: The study is conducted by 4 institutions: BCH (US clinical), WSU (bioanalytical core), USF (biostatistical core), and Heidelberg (iNTD), with support from the family advocacy group (SSADH Association). Recruitment goals were to study 20 patients on-site at BCH, 10 with iNTD, and 25 as a standard-of care cohort. Results: At this half-way point of this longitudinal study, 28 subjects have been recruited (57% female, mean 9 years, range 18 months–40 years). Epilepsy is present in half and increases in incidence and severity, as do psychiatric symptoms, in adolescence and adulthood. The average Full Scale IQ (FSIQ) was 53 (Verbal score of 56, Non Verbal score of 49), and half scored as having ASD. Although there was no correlation between gene variant and phenotypic severity, there were extreme cases of lowest functioning in one individual and highest in another that may have genotype-phenotype correlation. The most common EEG finding was mild background slowing with rare epileptiform activity, whereas high-density EEG and magnetoencephalography showed reduction in the gamma frequency band consistent with GABAergic dysfunction. MR spectroscopy showed elevations in the GABA/NAA ratio in all regions studied with no crossover between subjects and controls. Conclusions: The SSADH Natural History Study is providing a unique opportunity to study the complex pathophysiology longitudinally and derive electrophysiologic, neuroimaging, and laboratory data for correlation and to serve as biomarkers for clinical trials and prognostic assessments in this ultra-rare inherited disorder of GABA metabolism.

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Postmortem Analyses in a Patient With Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD): II. Histological, Lipid, and Gene Expression Outcomes in Regional Brain Tissue

Journal of Child Neurology ◽

10.1177/0883073820987742 ◽

2021 ◽

pp. 088307382098774

Author(s):

Dana C. Walters ◽

Regan Lawrence ◽

Trevor Kirby ◽

Jared T. Ahrendsen ◽

Matthew P. Anderson ◽

...

Keyword(s):

Gene Expression ◽

Dehydrogenase Deficiency ◽

Metabotropic Glutamate Receptor ◽

Total Phospholipid ◽

Null Mouse ◽

Succinic Semialdehyde ◽

Long Term Effects ◽

Succinic Semialdehyde Dehydrogenase ◽

Semialdehyde Dehydrogenase ◽

Succinic Semialdehyde Dehydrogenase Deficiency

This study has extended previous metabolic measures in postmortem tissues (frontal and parietal lobes, pons, cerebellum, hippocampus, and cerebral cortex) obtained from a 37-year-old male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) who expired from SUDEP (sudden unexplained death in epilepsy). Histopathologic characterization of fixed cortex and hippocampus revealed mild to moderate astrogliosis, especially in white matter. Analysis of total phospholipid mass in all sections of the patient revealed a 61% increase in cortex and 51% decrease in hippocampus as compared to (n = 2-4) approximately age-matched controls. Examination of mass and molar composition of major phospholipid classes showed decreases in phospholipids enriched in myelin, such as phosphatidylserine, sphingomyelin, and ethanolamine plasmalogen. Evaluation of gene expression (RT2 Profiler PCR Arrays, GABA, glutamate; Qiagen) revealed dysregulation in 14/15 GABAA receptor subunits in cerebellum, parietal, and frontal lobes with the most significant downregulation in ∊, θ, ρ1, and ρ2 subunits (7.7-9.9-fold). GABAB receptor subunits were largely unaffected, as were ionotropic glutamate receptors. The metabotropic glutamate receptor 6 was consistently downregulated (maximum 5.9-fold) as was the neurotransmitter transporter (GABA), member 13 (maximum 7.3-fold). For other genes, consistent dysregulation was seen for interleukin 1β (maximum downregulation 9.9-fold) and synuclein α (maximal upregulation 6.5-fold). Our data provide unique insight into SSADHD brain function, confirming astrogliosis and lipid abnormalities previously observed in the null mouse model while highlighting long-term effects on GABAergic/glutamatergic gene expression in this disorder.

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Magnetic Resonance Imaging (MRI) and Spectroscopy in Succinic Semialdehyde Dehydrogenase Deficiency

Journal of Child Neurology ◽

10.1177/0883073821991295 ◽

2021 ◽

pp. 088307382199129

Author(s):

Onur Afacan ◽

Edward Yang ◽

Alexander P. Lin ◽

Eduardo Coello ◽

Melissa L. DiBacco ◽

...

Keyword(s):

Magnetic Resonance Imaging ◽

Magnetic Resonance ◽

Mr Spectroscopy ◽

Succinic Semialdehyde ◽

Resonance Imaging ◽

Succinic Semialdehyde Dehydrogenase ◽

Semialdehyde Dehydrogenase ◽

Magnetic Resonance Imaging Mri ◽

Brain Gaba ◽

Ssadh Deficiency

Succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal recessive disorder of γ-aminobutyric acid (GABA) degradation, resulting in elevations of brain GABA and γ-hydroxybutyric acid (GHB). Previous magnetic resonance (MR) spectroscopy studies have shown increased levels of Glx in SSADH deficiency patients. Here in this work, we measure brain GABA in a large cohort of SSADH deficiency patients using advanced MR spectroscopy techniques that allow separation of GABA from overlapping metabolite peaks. We observed significant increases in GABA concentrations in SSADH deficiency patients for all 3 brain regions that were evaluated. Although GABA levels were higher in all 3 regions, each region had different patterns in terms of GABA changes with respect to age. We also report results from structural magnetic resonance imaging (MRI) of the same cohort compared with age-matched controls. We consistently observed signal hyperintensities in globus pallidus and cerebellar dentate nucleus.

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