Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

Glenn, Katie Frances; Smith, K. Shafer

doi:10.1128/jb.02380-14

Cited by 18 publications

(16 citation statements)

References 22 publications

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“…Phosphoketolase activity was measured using a hydroxymate assay that detects acetyl phosphate 35 , 57 , 58 . Briefly, 100 μL reactions in reaction buffer [30 mM potassium phosphate (pH 6.5), L- cysteine hydrochloride (2 mM), sodium fluoride (20 mM), sodium iodoacetate (10 mM), thiamine pyrophosphate (1 mM), and fructose 6-phosphate (30 mM) (Sigma)] were initiated by the addition of 25 μg total protein.…”

Section: Methodsmentioning

confidence: 99%

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach

Akberdin

Thompson

Hamilton

et al. 2018

Sci Rep

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Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20ZR an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization in M. alcaliphilum 20ZR. A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. The computational framework of C1-metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.

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Section: Methodsmentioning

confidence: 99%

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach

Akberdin

Thompson

Hamilton

et al. 2018

Sci Rep

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“…Phosphoketolase activity in whole-cell lysates prepared from logarithmically growing wild-type (20% CH 4 ), pPKTA, and pPKTB strains was measured using a hydroxymate assay that detects acetyl phosphate 26,27 . Briefly, cells (OD 600 = 1.0) were pelleted, resuspended in 50 mM potassium phosphate buffer (pH 6.5) and disrupted by sonication on ice.…”

Section: Phosphoketolase Enzyme Assaymentioning

confidence: 99%

Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Henard

Smith

Guarnieri

2017

Metabolic Engineering

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Microbial conversion of methane to high-value bio-based fuels, chemicals, and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense, encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. Given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.

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“…Evidence supporting the suggestion that AcP was indeed produced under these conditions included TLC of labeled cell extracts and enzymatic assays showing ATP generation from the cell lysates in the presence of highly purified acetate kinase. One alternative route for AcP generation that is present in a number of Gram-positive cocci with low GϩC contents, including some oral streptococci, is xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) (49). The Xfp enzyme catalyzes the conversion of F6P, in the presence of inorganic phosphate, to erythrose 4-phosphate and AcP.…”

Section: Figmentioning

confidence: 99%

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans

Kim

Ahn

Burne

2015

Appl Environ Microbiol

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In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ⌬ackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the ⌬pta or ⌬pta ⌬ackA mutant. The ⌬pta and ⌬pta ⌬ackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ⌬ackA strain. Surprisingly, when exposed to oxidative stress, the ⌬pta ⌬ackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ⌬ackA and ⌬pta ⌬ackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ⌬ackA and ⌬pta ⌬ackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans. Streptococcus mutans is a facultatively anaerobic, Gram-positive bacterium with fermentative metabolism. Human dental caries is associated with increased proportions of multiple acid-tolerant species in tooth biofilms, but S. mutans shows a particularly strong association with the initiation and progression of this common infectious disease (1, 2). The pathogenic potential of S. mutans is highly dependent on its ability to form biofilms, to use a variety of carbohydrates to produce organic acids that dissolve tooth mineral, and to tolerate stresses commonly encountered in oral biofilms. In particular, tolerance of a variety of reactive oxygen species (ROS), including superoxide ions and hydrogen peroxide, is considered critical for S. mutans to overcome antagonism by oral commensals and host defenses (3, 4).S. mutans has a partial tricarboxylic acid (TCA) cycle and lacks cytochromes, so the primary route for ATP generation by this organism is through the Embden-Meyerhof-Parnas pathway (4-7). Under anaerobic conditions and with growth in the presence of an excess of a preferred carbohydrate, the pyruvate generated by glycolysis is acted on by lactate dehydrogenase (LDH), which is allosterically activated by fructose-1,6-bisphosphate (F-1,6-BP), to produce lactate and regenerate NAD (4, 8). If carbohydrate is limiting, S. mutans produces a pyruvate formate lyase enzyme, which converts pyruvate to acetyl coenzyme A (acetyl-CoA) and formate (Fig. 1). However, the pyruvate formate lyase (PFL) enzyme of S. mutans is inactivated by oxygen, so under aerobic conditions and if catabolite repression is alleviated (M. Watts and R. A....

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Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

Cited by 18 publications

References 22 publications

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach

Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans

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