Catalytic properties of wheat phytase that favorably degrades long-chain inorganic polyphosphate

Abstract An active site normally locates inside of enzymes, substrates should go through the tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450 Bsβ HI (Q85H/ V170I) derived from hydroxylase P450 Bsβ from Bacillus subtilis was chosen as study model, which is reported as a potential decarbonylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarbonylase activity of P450 Bsβ HI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.

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Role of inorganic polyphosphate in mammalian cells: from signal transduction and mitochondrial metabolism to cell death

Biochemical Society Transactions ◽

10.1042/bst20150223 ◽

2016 ◽

Vol 44 (1) ◽

pp. 40-45 ◽

Cited By ~ 30

Author(s):

Plamena R. Angelova ◽

Artyom Y. Baev ◽

Alexey V. Berezhnov ◽

Andrey Y. Abramov

Keyword(s):

Signal Transduction ◽

Cell Death ◽

Mammalian Cells ◽

High Energy ◽

Mitochondrial Metabolism ◽

Calcium Handling ◽

Calcium Signal ◽

Inorganic Polyphosphate ◽

Long Chain

Inorganic polyphosphate (polyP) is a polymer compromised of linearly arranged orthophosphate units that are linked through high-energy phosphoanhydride bonds. The chain length of this polymer varies from five to several thousand orthophosphates. PolyP is distributed in the most of the living organisms and plays multiple functions in mammalian cells, it is important for blood coagulation, cancer, calcium precipitation, immune response and many others. Essential role of polyP is shown for mitochondria, from implication into energy metabolism and mitochondrial calcium handling to activation of permeability transition pore (PTP) and cell death. PolyP is a gliotransmitter which transmits the signal in astrocytes via activation of P2Y1 receptors and stimulation of phospholipase C. PolyP-induced calcium signal in astrocytes can be stimulated by different lengths of this polymer but only long chain polyP induces mitochondrial depolarization by inhibition of respiration and opening of the PTP. It leads to induction of astrocytic cell death which can be prevented by inhibition of PTP with cyclosporine A. Thus, medium- and short-length polyP plays role in signal transduction and mitochondrial metabolism of astrocytes and long chain of this polymer can be toxic for the cells.

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Deficiency of the Novel Exopolyphosphatase Rv1026/PPX2 Leads to Metabolic Downshift and Altered Cell Wall Permeability in Mycobacterium tuberculosis

mBio ◽

10.1128/mbio.02428-14 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 48

Author(s):

Yu-Min Chuang ◽

Nirmalya Bandyopadhyay ◽

Dalin Rifat ◽

Harvey Rubin ◽

Joel S. Bader ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Stringent Response ◽

Inorganic Polyphosphate ◽

Content Type ◽

P Accumulation ◽

Wall Permeability ◽

Altered Cell ◽

Cell Wall Permeability ◽

Long Chain

ABSTRACTMycobacterium tuberculosiscan persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here thatM. tuberculosispoly(P) accumulation is temporally linked to bacillary growth restriction. We also identifyM. tuberculosisRv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression ofRv1026(ppx2), we found thatM. tuberculosispoly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, theppx2knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of theppx2knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role inM. tuberculosisgrowth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability.IMPORTANCEThe stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediateMycobacterium tuberculosispersistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in theppx2gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis.

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Tunnel Engineering for Modulating The Substrate Preference in Decarbonylase P450BsβHI

10.21203/rs.3.rs-132813/v1 ◽

2020 ◽

Author(s):

Shuaiqi Meng ◽

Ruipeng An ◽

Zhongyu Li ◽

Ulrich Schwaneberg ◽

Yu Ji ◽

...

Keyword(s):

Active Site ◽

Catalytic Properties ◽

Substrate Preference ◽

Decarboxylase Activity ◽

Long Chain Fatty Acids ◽

Pentadecanoic Acid ◽

Tunnel Engineering ◽

Engineering Strategy ◽

Powerful Strategy ◽

Long Chain

Abstract An active site normally locates inside of enzymes, substrates should go through the tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450BsβHI (Q85H/ V170I) derived from hydroxylase P450Bsβ from Bacillus subtilis was chosen as study model, which is reported as a potential decarbonylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarbonylase activity of P450BsβHI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.

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The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.4812-4819.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 4812-4819 ◽

Cited By ~ 21

Author(s):

Silvia T. Cardona ◽

Francisco P. Chávez ◽

Carlos A. Jerez

Keyword(s):

Sulfolobus Solfataricus ◽

Open Reading Frame ◽

Inorganic Polyphosphate ◽

Reading Frame ◽

Highly Active ◽

Calculated Mass ◽

Polyphosphate Metabolism ◽

Long Chain

ABSTRACT Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.

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Tunnel engineering for modulating the substrate preference in cytochrome P450BsβHI

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00379-1 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Shuaiqi Meng ◽

Ruipeng An ◽

Zhongyu Li ◽

Ulrich Schwaneberg ◽

Yu Ji ◽

...

Keyword(s):

Active Site ◽

Catalytic Properties ◽

Substrate Preference ◽

Decarboxylase Activity ◽

Long Chain Fatty Acids ◽

Pentadecanoic Acid ◽

Tunnel Engineering ◽

Engineering Strategy ◽

Powerful Strategy ◽

Long Chain

AbstractAn active site is normally located inside enzymes, hence substrates should go through a tunnel to access the active site. Tunnel engineering is a powerful strategy for refining the catalytic properties of enzymes. Here, P450BsβHI (Q85H/V170I) derived from hydroxylase P450Bsβ from Bacillus subtilis was chosen as the study model, which is reported as a potential decarboxylase. However, this enzyme showed low decarboxylase activity towards long-chain fatty acids. Here, a tunnel engineering campaign was performed for modulating the substrate preference and improving the decarboxylation activity of P450BsβHI. The finally obtained BsβHI-F79A variant had a 15.2-fold improved conversion for palmitic acid; BsβHI-F173V variant had a 3.9-fold improved conversion for pentadecanoic acid. The study demonstrates how the substrate preference can be modulated by tunnel engineering strategy.

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Polymer Morphology Revealed by Staining Techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098204 ◽

1981 ◽

Vol 39 ◽

pp. 340-341

Author(s):

A. C. Reimschuessel ◽

V. Kramer

Keyword(s):

Polymer Melt ◽

Nylon 6 ◽

Morphological Features ◽

Negative Staining ◽

Polymer Morphology ◽

Crystallizable Polymer ◽

Staining Techniques ◽

Long Chain

Staining techniques can be used for either the identification of different polymers or for the differentiation of specific morphological domains within a given polymer. To reveal morphological features in nylon 6, we choose a technique based upon diffusion of the staining agent into accessible regions of the polymer.When a crystallizable polymer - such as nylon 6 - is cooled from the melt, lamellae form by chainfolding of the crystallizing long chain macromolecules. The regions between adjacent lamellae represent the less ordered amorphous domains into which stain can diffuse. In this process the lamellae will be “outlined” by the dense stain, giving rise to contrast comparable to that obtained by “negative” staining techniques.If the cooling of the polymer melt proceeds relatively slowly - as in molding operations - the lamellae are usually arranged in a radial manner. This morphology is referred to as spherulitic.

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