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 milliunits͞mg and phosphate ester hydrolysis with specific activities of 41-61 units͞ mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H 2. 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.U nlike the other major elements in living organisms, P is commonly considered to be a redox conservative element. Accordingly, the P centers found in the vast majority of biological intermediates, including inorganic phosphate (P i ), organic phosphate esters, and phosphoanhydrides, are fully oxidized (ϩ5 valence state). Nevertheless, it is now clear, although not widely appreciated, that many organisms are capable of metabolizing reduced P compounds, indicating that P redox reactions are biochemically possible. A wide array of prokaryotic (and some eukaryotic) organisms can synthesize or degrade reduced P compounds (1, 2). Moreover, the widespread occurrence of this trait strongly suggests that a selective advantage is conferred on organisms with the ability to metabolize reduced P compounds. Examination of reduced P metabolism has revealed a wealth of unusual biology and biochemistry. The bacterium Desulfotignum phosphitoxidans gains energy for growth by oxidation of phosphite (Pt) coupled to either sulfate reduction or acetogenesis while fixing CO 2 as its sole C source (3, 4). Many other organisms can use reduced P compounds as sole P sources (5-8). For example, Pseudomonas stutzeri is capable of oxidizing hypophosphite (P valence, ϩ1) to phosphate by means of a Pt (P valence, ϩ3) intermediate (9). The following two enzymes catalyze these reactions: 2-oxoglutarate͞hypophosphite dioxygenase catalyzes the oxidation of hypo-Pt to Pt (10), and Pt͞NAD oxidoreductase catalyzes the oxidation of Pt to phosphate (11). The latter reaction is the most thermodynamically favorable reduction of NAD that is known, and this trait makes this enzyme particularly useful as a cofactor-regenerating catalyst for enzyme-based synthetic strategies (12). P biochemistry can be found also in more familiar organisms, such as Escherichia coli. As shown below, E. coli has two pathways for the oxidation of Pt. Our examination of these pathways demonstrates that thoroughly characterized biochemist...

An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily

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AcpA Is aFrancisellaAcid Phosphatase That Affects Intramacrophage Survival and Virulence

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Purification and Properties of Alkaline Phosphatase fromBacillus Cereus

“…Alkaline phosphatases have been identified in a wide variety of organisms, including bacteria (Escherichia coli, Bacillus species, Mycobacterium smegmatis, Thermotoga maritime, Haloarcula marismortui) and humans (3,15,12,13,17,21,22). Although the actual purpose of the enzyme is still not fully understood, the simple hypothesis, that it is a means for the bacteria to generate free phosphate groups for uptake and use (20).…”