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Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium, Microlunatus phosphovorus.

ABSTRACT: ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.

SUBMITTER: Tanaka S

PROVIDER: S-EPMC193765 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium, Microlunatus phosphovorus.

Tanaka Shotaro S Lee Sun-Og SO Hamaoka Kazuhiro K Kato Junichi J Takiguchi Noboru N Nakamura Kazunori K Ohtake Hisao H Kuroda Akio A

Journal of bacteriology 20030901 18

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis. ...[more]

PMID: 12949120

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Project description:Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm ), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

Project description:Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Dataset Information

Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium, Microlunatus phosphovorus.

Publications

Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium, Microlunatus phosphovorus.

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