Stereochemical probe for the mechanism of phosphorus-carbon bond cleavage catalyzed by the Bacillus cereus phosphonoacetaldehyde hydrolase

Lee, Sheng Lian; Hepburn, Timothy W.; Swartz, W. H.; Ammon, Herman L.; Mariano, Patrick S.; Dunaway‐Mariano, Debra

doi:10.1021/ja00045a003

Cited by 34 publications

(21 citation statements)

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“…AEPn is converted to phosphonoacetaldehyde by an AEPnspecific transaminase, and phosphonoacetaldehyde is subsequently converted to acetaldehyde and P i by phosphonoacetaldehyde hydrolase (trivial name, phosphonatase). Enzymes carrying out both of these steps have been characterized for Bacillus cereus (26) and Pseudomonas aeruginosa (12,23). Uptake systems specific for AEPn have also been demonstrated for these bacteria (22,39), consistent with breakdown occurring in the cytoplasm.…”

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confidence: 84%

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

et al. 1995

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Two pathways exist for cleavage of the carbon-phosphorus (C-P) bond of phosphonates, the C-P lyase and the phosphonatase pathways. It was previously demonstrated that Escherichia coli carries genes (named phn) only for the C-P lyase pathway and that Enterobacter aerogenes carries genes for both pathways (K.-S. Lee, W. W. Metcalf, and B. L. Wanner, J. Bacteriol. 174:2501-2510, 1992). In contrast, here it is shown that Salmonella typhimurium LT2 carries genes only for the phosphonatase pathway. Genes for the S. typhimurium phosphonatase pathway were cloned by complementation of E. coli ⌬phn mutants. Genes for these pathways were proven not to be homologous and to lie in different chromosomal regions. The S. typhimurium phn locus lies near 10 min; the E. coli phn locus lies near 93 min. The S. typhimurium phn gene cluster is about 7.2 kb in length and, on the basis of gene fusion analysis, appears to consist of two (or more) genes or operons that are divergently transcribed. Like that of the E. coli phn locus, the expression of the S. typhimurium phn locus is activated under conditions of P i limitation and is subject to Pho regulon control. This was shown both by complementation of the appropriate E. coli mutants and by the construction of S. typhimurium mutants with lesions in the phoB and pst loci, which are required for activation and inhibition of Pho regulon gene expression, respectively. Complementation studies indicate that the S. typhimurium phn locus probably includes genes both for phosphonate transport and for catalysis of C-P bond cleavage.Phosphonates (Pn) are a large class of molecules containing a direct carbon-phosphorus (C-P) bond in place of the more familiar carbon-oxygen-phosphorus bond of phosphoesters. Although Pn do not exist in all organisms, they do exist in many different ones. Pn have been found in organisms as diverse as the intracellular procaryote Bdellovibrio species, the filamentous bacteria streptomycetes, the protozoans Tetrahymena and Trypanosoma spp., mollusks, insects, and others. In these organisms, Pn have been isolated as constituents of glycolipids, glycoproteins, polysaccharides, or phosphonolipids. In Bacteroides fragilis, Pn were identified as components of capsular polysaccharide (6); in streptomycetes, Pn are synthesized as antibiotics such as fosfomycin (25); in Tetrahymena spp., Pn exist as phosphonolipids (19); in Trypanosoma cruzi, Pn are components of lipopeptidophosphoglycan (the major cell surface glycoconjugate [10]); in a sea anemone, Pn are the major P compounds (38); and in a locus, a Pn is the principal P compound of hemolymph (20). Yet, in spite of their widespread natural occurrence, the biological role of natural Pn is poorly understood (17). In phosphonolipids, 2-aminoethylphosphonate (AEPn) exists in place of its analog ethanolamine phosphate.No doubt because of the abundance of PN in nature, bacteria have acquired pathways for Pn breakdown. Bacteria capable of breaking the C-P bond may use Pn as a sole P source, for which two pathways exist, the C-P l...

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confidence: 84%

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

et al. 1995

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“…In view of the known stereochemistry of the phosphonatase reaction (26), retention of configuration at phosphorus and hydrolysis of the acyl-phosphoenzyme formed must necessarily occur at phosphorus. Indeed, the sarcoplasmic ATPase, which is known to catalyze the hydrolysis of ATP via formation of an acyl-phosphoenzyme intermediate (Asp351) (52), does so with overall retention of stereochemistry at the γ-phosphoryl phosphorus (53).…”

Section: Possible Link Between the Conserved Aspartate Of Superfamilymentioning

confidence: 99%

“…The formation of phosphate from Pald occurs, in the second partial reaction, with overall retention of stereochemistry at phosphorus (as determined using chiral [ 17 O, 18 O]-thiophosphonoacetaldehyde as the substrate), suggesting that the phosphoryl group is first transferred to an active nucleophile before it is intercepted by solvent water (26). The formation of a phosphoenzyme during the course of phosphoryl transfer is a common mechanistic theme among phosphotransferases, particularly among phosphatases.…”

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Insights into the Mechanism of Catalysis by the P−C Bond-Cleaving Enzyme Phosphonoacetaldehyde Hydrolase Derived from Gene Sequence Analysis and Mutagenesis

et al. 1998

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Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli. The kinetic properties of the purified, recombinant phosphonatases were determined. The Schiff base mechanism known to operate in the B. cereus enzyme was verified for the S. typhimurium enzyme by phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein sequence inferred from the B. cereus phosphonatase gene was determined, and this sequence was used along with that from the S. typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis. On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereus phosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.Phosphonates constitute a class of naturally occurring organophosphorus compounds which differ in structure from the more predominate phosphate esters by the direct linkage of phosphorus to carbon (1-3). The P-C bond is resistant to acid-and base-catalyzed hydrolysis as well as to enzymes which catalyze the cleavage of P-O-C bonds in phosphate esters. Their similarity to phosphate ester structure, coupled with their stability, gives phosphonates a variety of biological properties which we have seen exploited in both natural products (e.g., antibiotics and phosphatase-resistant membrane components) and synthetics (e.g., insecticides, herbicides, and pharmaceuticals). Although phosphonates have been found in numerous organisms ranging from bacteria to mammals, active synthesis of phosphonates has thus far been demonstrated in only certain bacteria (4-6), a protozoan Tetrahymena pyriformis (7,8), and a mollusk Mytilus edulis (9). Phosphonate degradative pathways, on the other hand, have been demonstrated only in bacteria (1-3).It has been speculated that phosphonate natural products are a vestige of an earlier era on earth when reduced forms of organophosphorus compounds predominated over organophosphates (1-3, 10). If this is true, then the phosphonohydrolases are likely to have evolved long ago to allow the cycling of phosphorus between phosphonates and phosphates. Presently, three P-C bond-cleaving enzymes are known. The C-P lyase, which has a very broad substrate specificity, cleaves the C-P bond homolytically, yet ultimately produces inorganic phosphate and the corresponding hydrocarbon...

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“…Phosphonates, which contain a P-C bond, differ in chemical stability from the more common phosphate monoesters, which possess a P-O-C bond linkage [5][6][7]. The catalytic pathways for their hydrolysis also differ [8,9]. We were thus intrigued by the discovery that phosphonatase is a member of the haloacid dehalogenase superfamily (HADSF) [10], a family predominantly comprised of phosphatases.…”

Section: Introductionmentioning

confidence: 99%

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon bond cleavage

Lahiri

Zhang

Dunaway‐Mariano

et al. 2006

Bioorganic Chemistry

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Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the C-P bond in phosphonoacetaldehyde (Pald) via formation of a bicovalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 Å X-ray crystal structure of the mutant Lys53Arg complexed with Mg +2 and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain-core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH 4 in the presence of Pald was determined at 2.4 Å resolution to reveal Nε-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C(2) of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain Nε-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bicovalent enzyme intermediate. Because the enzyme residues proposed to play a key role in P-C bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic P-C bond cleavage.

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Stereochemical probe for the mechanism of phosphorus-carbon bond cleavage catalyzed by the Bacillus cereus phosphonoacetaldehyde hydrolase

Cited by 34 publications

References 4 publications

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

Insights into the Mechanism of Catalysis by the P−C Bond-Cleaving Enzyme Phosphonoacetaldehyde Hydrolase Derived from Gene Sequence Analysis and Mutagenesis

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon bond cleavage

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