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A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

ABSTRACT: Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62-242 milliunits/mg and phosphate ester hydrolysis with specific activities of 41-61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H2. Thus, BAP is a unique Pt-dependent, H2-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.

SUBMITTER: Yang K

PROVIDER: S-EPMC419532 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

Yang Kechao K Metcalf William W WW

Proceedings of the National Academy of Sciences of the United States of America 20040517 21

Genetic analysis indicates that Escherichia coli possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene phn operon, which encodes the enzyme C-P lyase. The other pathway depends on the phoA locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the phoA-dependent pathway. This conclusion is supported by purification and biochemical characterizat ...[more]

PMID: 15148399

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Project description:1. By digitonin lysis of penicillin spheroplasts of Escherichia coli a particulate fraction P(1) was previously obtained that supported the sustained synthesis of alkaline phosphatase when supplied with amino acids, nucleotide triphosphates and other cofactors. This P(1) fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg(2+), yielded the P(1)(S) fraction, consisting of integrated particulate subcellular particles containing DNA and RNA. 2. The P(1)(S) fraction from E. coli K10 wild type (R(+) (1)R(+) (2)P(+)) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in vitro. The synthesis occurred in phases. The first was followed by a lag, and then there was a linear rapid phase that continued for at least 3hr. Actinomycin D inhibited the appearance of the second phase. It was concluded that the particles are programmed to synthesize enzyme even when prepared from repressed cells, and therefore that synthesis of the specific messenger RNA for alkaline phosphatase in vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. 3. Phosphate inhibited synthesis of enzyme to the same extent with the P(1)(S) fractions of two constitutive strains as with the P(1)(S) fraction of the wild-type strain. 4. Inorganic phosphate inhibited amino acid incorporation with the P(1)(S) fraction and also inhibited enzyme synthesis in vitro. The effect on amino acid incorporation could be partially overcome by adding Mn(2+) to the incubation mixtures. However, Mn(2+) inhibited the synthesis of alkaline phosphatase. Also, inhibition of the incorporation of [(32)P]CTP into RNA was overcome by Mn(2+). The effect of phosphate on amino acid uptake was most probably due to a phosphorolysis of RNA by polynucleotide phosphorylase, also present in the P(1)(S) fraction. This phosphorolysis may be responsible for the instability of messenger RNA in vitro and in vivo. 5. Phosphate also specifically inhibited the formation of alkaline phosphatase, since it did not affect markedly the induced formation of beta-galactosidase by the same P(1)(S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn(2+)-dependent reaction. It is suggested that the repression of the synthesis of alkaline phosphatase in vivo in the wild-type strain was the sum of these two effects.

Dataset Information

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

Publications

A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

Similar Datasets

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