Phosphonoacetaldehyde Hydrolase Research Articles

Laundry Isolate Delftia sp. UBM14 Capable of Biodegrading Industrially Relevant Aminophosphonates.

Phosphonates such as ethylenediaminetetra (methylenephosphonic acid) (EDTMP) and aminotris (methylenephosphonic acid) (ATMP) are used every day in water treatment processes or in household products. Their consumption is still increasing, regardless of the debates on their environmental impact. Here, the microbial characterisation and determination of the biodegradation potential of selected industrially relevant phosphonates for the isolate Delftia sp. UMB14 is reported. The opportunistic strain was isolated from a biofilm that was derived from a conventional washing machine using conventional detergents containing phosphonates. In antimicrobial susceptibility testing, the strain was only susceptible to sulfonamide, tetracycline, and chloramphenicol. Physiological and biochemical characteristics were determined using the BIOLOG EcoPlate assay. Most importantly, the strain was shown to convert D-malic acid and D-mannitol, as confirmed for strains of Delftia lacustris, and thus the new isolate could be closely related. Biodegradation tests with different phosphonates showed that the strain preferentially degrades ATMP and EDTMP but does not degrade glyphosate (GS) and amino (methylphosphonic acid) (AMPA). A specific gene amplification confirmed the presence of phnX (phosphonoacetaldehyde hydrolase) and the absence of PhnJ (the gene for the core component of C-P lyase). The presence of PhnCDE is strongly suggested for the strain, as it is common in Delftia lacustris species.

Glyphosate-Induced Phosphonatase Operons in Soil Bacteria of the Genus Achromobacter.

Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.

2-Aminoethylphosphonate Utilization by the Cold-Adapted Geomyces pannorum P11 Strain

Cold-adapted strain of Geomyces pannorum P11 was found to mineralize of phosphorus–carbon bond-containing compound—2-aminoethylphosphonic acid (2-AEP, ciliatine). The biodegradation process proceeded in the phosphate-independent manner. Ciliatine-metabolizing enzymes' activity was detectable in cell-free extracts prepared from psychrophilic G. pannorum pregrown on 4 mM 2-AEP. Phosphonoacetaldehyde hydrolase (phosphonatase) activity in a partially purified extract was demonstrated at 10 °C.

Towards understanding phosphonoacetaldehyde hydrolase: an alternative mechanism involving proton transfer that triggers P–C bond cleavage

The theoretical QM/MM study of the reaction catalysed by phosphonoacetaldehyde hydrolase indicates a possible alternative mechanism of the P-C bond cleavage: as opposed to the mechanism proposed earlier and that involved formation of a covalently bound intermediate (Schiff-base), in the new mechanism, the bond breaking process is facilitated by proton transfer from catalytic lysine residue to the substrate.

Theoretical insights into catalysis by phosphonoacetaldehyde hydrolase§

Phosphonatase catalyses P–C bond cleavage in phosphonoacetaldehyde molecules. According to the experimental results this reaction is initiated by Schiff base formation, which facilitates nucleophilic attack on phosphorus. In this paper a preliminary theoretical description of Schiff base formation, P–C bond cleavage and phosphate release is presented, based on quantum chemical calculations at the DFT level of theory, which is probably the first theoretical study of a reaction catalyzed by phosphonatase. Findings from these calculations support the mechanism of imine formation reported in the literature and show that different pathways of phosphonyl transfer can be applied in the active site of phosphonatase. §This contribution is dedicated to Professor Andrzej J. Sadlej on the occasion of his 65th birthday.

Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily.

The 2-haloalkanoic acid dehalogenase (HAD) family, which contains both carbon and phosphoryl transferases, is one of the largest known enzyme superfamilies. HAD members conserve an alpha,beta-core domain that frames the four-loop active-site platform. Each loop contributes one or more catalytic groups, which function in mediating the core chemistry (i.e., group transfer). In this paper, we provide evidence that the number of carboxylate residues on loop 4 and their positions (stations) on the loop are determinants, and therefore reliable sequence markers, for metal ion activation among HAD family members. Using this predictor, we conclude that the vast majority of the HAD members utilize a metal cofactor. Analysis of the minimum requirements for metal cofactor binding was carried out using Mg(II)-activated Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) as an experimental model for metal-activated HAD members. Mg(II) binding occurs via ligation to the loop 1 Asp12 carboxylate and Thr14 backbone carbonyl and to the loop 4 Asp186 carboxylate. The loop 4 Asp190 forms a hydrogen bond to the Mg(II) water ligand. X-ray structure determination of the D12A mutant in the presence of the substrate phosphonoacetaldehyde showed that replacement of the loop 1 Asp, common to all HAD family members, with Ala shifts the position of Mg(II), thereby allowing innersphere coordination to Asp190 and causing a shift in the position of the substrate. Kinetic analysis of the loop 4 mutants showed that Asp186 is essential to cofactor binding while Asp190 simply enhances it. Within the phosphonatase subfamily, Asp186 is stringently conserved, while either position 185 or position 190 is used to position the second loop 4 Asp residue. Retention of a high level of catalytic activity in the G185D/D190G phosphonatase mutant demonstrated the plasticity of the metal binding loop, reflected in the variety of combinations in positioning of two or three Asp residues along the seven-residue motif of the 2700 potential HAD sequences that were examined.

Analysis of the substrate specificity loop of the HAD superfamily cap domain.

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.

X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) to form orthophosphate and acetaldehyde. The reaction proceeds via a Schiff-base intermediate formed between Lys-53 and the Pald carbonyl. The x-ray crystal structures of the wild-type phosphonatase complexed with Mg(II) alone or with Mg(II) plus vinylsulfonate (a phosphonoethylenamine analog) were determined to 2.8 and 2.4 A, respectively. These structures were used to determine the identity and positions of active site residues surrounding the Lys-53 ammonium group and the Pald carbonyl. These include Cys-22, His-56, Tyr-128, and Met-49. Site-directed mutagenesis was then employed to determine whether or not these groups participate in catalysis. Based on rate contributions, Tyr-128 and Cys-22 were eliminated as potential catalytic groups. The Lys-53 epsilon-amino group, positioned for reaction with the Pald carbonyl, forms a hydrogen bond with water 120. Water 120 is also within hydrogen bond distance of an imidazole nitrogen of His-56 and the sulfur atom of Met-49. Kinetic constants for mutants indicated that His-56 (1000-fold reduction in k(cat)/K(m) upon Ala substitution) and Met-49 (17,000-fold reduction in k(cat)/K(m) upon Leu substitution) function in catalysis of Schiff-base formation. Based on these results, it is proposed that a network of hydrogen bonds among Lys-53, water 120, His-56, and Met-49 facilitate proton transfer from Lys-53 to the carbinolamine intermediate. Comparison of the vinylsulfonate complex versus unliganded structures indicated that association of the cap and core domains is essential for the positioning of the Lys-53 for attack at the Pald carbonyl and that substrate binding at the core domain stabilizes cap domain binding.

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

Phosphonoacetaldehyde (Pald) is formed in a variety of biosynthetic pathways leading to natural phosphonates and is an intermediate in the degradation pathway of the natural product 2-aminoethylphosphonate. To facilitate the investigation of the enzymes catalyzing these pathways, a method for the synthesis of radiolabeled Pald was developed. The enzyme pyruvate phosphate dikinase was used to prepare phosphoenolpyruvate (PEP) from pyruvate, adenosine triphosphate (ATP), and orthophosphate. Then PEP was converted to phosphonopyruvate (Ppyr) with PEP mutase and then to Pald with Ppyr decarboxylase. By using [β- 32P]ATP or [2- 14C]pyruvate as precursor, [ 32P]Pald or [1- 14C]Pald was obtained, respectively. The utilization of the synthetic, radiolabeled Pald as a probe of enzyme mechanism was demonstrated with the enzyme phosphonoacetaldehyde hydrolase (trivial name phosphonatase). The single turnover time course for the formation and consumption of radiolabeled covalent enzyme species evidenced a kinetically competent covalent intermediate.

The Purification and Characterization of Phosphonopyruvate Hydrolase, a Novel Carbon-Phosphorus Bond Cleavage Enzyme from Variovorax sp. Pal2

Phosphonopyruvate hydrolase, a novel bacterial carbon-phosphorus bond cleavage enzyme, was purified to homogeneity by a series of chromatographic steps from cell extracts of a newly isolated environmental strain of Variovorax sp. Pal2. The enzyme was inducible in the presence of phosphonoalanine or phosphonopyruvate; unusually, its expression was independent of the phosphate status of the cell. The native enzyme had a molecular mass of 63 kDa with a subunit mass of 31.2 kDa. Activity of purified phosphonopyruvate hydrolase was Co2+-dependent and showed a pH optimum of 6.7-7.0. The enzyme had a Km of 0.53 mm for its sole substrate, phosphonopyruvate, and was inhibited by the analogues phosphonoformic acid, 3-phosphonopropionic acid, and hydroxymethylphosphonic acid. The nucleotide sequence of the phosphonopyruvate hydrolase structural gene indicated that it is a member of the phosphoenolpyruvate phosphomutase/isocitrate lyase superfamily with 41% identity at the amino acid level to the carbon-to-phosphorus bond-forming enzyme phosphoenolpyruvate phosphomutase from Tetrahymena pyriformis. Thus its apparently ancient evolutionary origins differ from those of each of the two carbon-phosphorus hydrolases that have been reported previously; phosphonoacetaldehyde hydrolase is a member of the haloacetate dehalogenase family, whereas phosphonoacetate hydrolase belongs to the alkaline phosphatase superfamily of zinc-dependent hydrolases. Phosphonopyruvate hydrolase is likely to be of considerable significance in global phosphorus cycling, because phosphonopyruvate is known to be a key intermediate in the formation of all naturally occurring compounds that contain the carbon-phosphorus bond.

Kinetic evidence for a substrate-induced fit in phosphonoacetaldehyde hydrolase catalysis.

Phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) via a Schiff base intermediate formed with Lys53. A single turnover requires binding of Pald to the active site of the core domain, closure of the cap domain containing the Lys53 over the core domain, and dissociation of the products following catalysis. The ligand binding and dissociation steps occur from the "open conformer" (domains are separated and the active site is solvent-exposed), while catalysis occurs from the "closed conformer" (domains are bound together and the active site is sequestered from solvent). To test the hypothesis that bound substrate stabilizes the closed conformer, thus facilitating catalysis, the rates of chemical modification of Lys53 in the presence and absence of inert substrate and/or product analogues were compared. Acetylation of Lys53 with 2,4-dinitrophenylacetate (DNPA) resulted in the loss of enzyme activity. The pseudo-first-order rate constant for inactivation varied with pH. The pH profile of inactivation is consistent with a pK(a) of 9.3 for Lys53. The inhibitors tungstate and vinyl sulfonate, which are known to bind to active site residues comprising the core domain, protected Lys53 from acetylation. These results are consistent with a dynamic equilibrium between the open and closed conformations of phosphonatase and the hypothesis that ligand binding stabilizes the closed conformation required for catalytic turnover.

The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.

Organophosphonate metabolism by a moderately halophilic bacterial isolate

A Gram-negative halophile isolated from soil beneath a road gritting salt pile grew optimally at 10% (w/v) NaCl and was shown most likely to be Chromohalobacter marismortui or Pseudomonas beijerinckii on the basis of 16S rRNA analysis. The strain utilised phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutyl-, methyl- and ethyl-phosphonates as phosphorus sources for growth. Differences were observed in the growth rate on different phosphonates and the range of phosphonates utilised at elevated NaCl concentrations, possibly as a result of differentially-induced transport mechanisms. An assay of cell-free extracts of 2-aminoethylphosphonate (2AEP) grown cells showed no detectable 2AEP:pyruvate aminotransferase or phosphonoacetaldehyde hydrolase activity.

Organophosphonate metabolism by a moderately halophilic bacterial isolate.

A Gram-negative halophile isolated from soil beneath a road gritting salt pile grew optimally at 10% (w/v) NaCl and was shown most likely to be Chromohalobacter marismortui or Pseudomonas beijerinckii on the basis of 16S rRNA analysis. The strain utilised phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutyl-, methyl- and ethyl-phosphonates as phosphorus sources for growth. Differences were observed in the growth rate on different phosphonates and the range of phosphonates utilised at elevated NaCl concentrations, possibly as a result of differentially-induced transport mechanisms. An assay of cell-free extracts of 2-aminoethylphosphonate (2AEP) grown cells showed no detectable 2AEP:pyruvate aminotransferase or phosphonoacetaldehyde hydrolase activity.

Crystallization and preliminary crystallographic analysis of phosphonoacetaldehyde hydrolase

Phosphonoacetaldehyde hydrolase, a C-P bond-cleaving enzyme which utilizes an unusual bicovalent catalytic strategy, has been crystallized by the hanging-drop vapor-diffusion method using PEG 4000 as the precipitant. The crystals belong to the monoclinic system and belong to space group C2, with unit-cell parameters a = 210.5, b = 45.5, c = 64.7 A, beta = 105.0 degrees. The asymmetric unit contains a dimer related by a non-crystallographic dyad. In addition to a 2.7 A native data set, the following data sets have been collected: a 2.4 A data set from crystals complexed with the intermediate analog vinyl sulfonate, a 3.0 A three-wavelength MAD data set from crystals complexed with the product analog WO(4)(2-), as well as several heavy-atom data sets to 3.0 A or better, of which only three have proven useful for MIR calculations. Examination of the native Patterson map revealed NCS that made previously uninterpretable derivative data useful. Independent phase sets were first calculated and refined for the MAD and MIR experiments separately and were then combined. The combined phase set was further improved by solvent flattening, histogram matching and NCS averaging. Interpretation of the resulting electron-density map is currently under way.

Phosphate Starvation-independent 2-Aminoethylphosphonic Acid Biodegradation in a Newly Isolated Strain of Pseudomonas putida, NG2

A strain of Pseudomonas putida that utilized the biogenic organophosphonate 2-aminoethylphosphonic acid as sole carbon and energy, nitrogen and phosphorus source contained 2-aminoethylphosphonic acid: pyruvate aminotransferase and phosphonoacetaldehyde hydrolase (phosphonatase) activities which were inducible by the presence of 2-aminoethylphosphonic acid in the culture medium, regardless of the phosphate status of the cells. Neither of these activities were induced in their phosphate-free or phosphate-replete medium in the absence of 2-aminoethylphosphonic acid. Alkaline phosphatase activity was induced in phosphate limited medium, however, indicating a phosphate-starvation inducible response. In Enterobacter aerogenes IFO 12010, 2-aminoethylphosphonate: pyruvate aminotransferase and phosphonatase activities were induced only when cells were both phosphate limited and supplied with 2-aminoethylphosphonic acid as sole phosphorus source for growth. Neither enzyme activity was induced in phosphate-replete medium, or in medium where both 2-aminoethylphosphonic acid and inorganic phosphate were supplied as sources of phosphorus. The results point to the presence of a substrate inducible 2-aminoethylphosphonic acid biodegradation pathway in the isolated strain of Pseudomonas putida. Uniquely, therefore, the pathway is not under pho regulon control in this strain.

First characterization of the phosphonoacetaldehyde hydrolase gene of Pseudomonas aeruginosa

The phnX gene encoding the phosphonoacetaldehyde hydrolase (phosphonatase) from the Gram-negative bacterium Pseudomonas aeruginosa A237 has been cloned and its sequence determined. The open reading frame consists of 825 nucleotides specifying a protein of 275 amino acid residues corresponding to a predicted molecular weight of 29 929. The deduced amino acid sequence of PhnX did not share significant amino acid sequence similarity with any other polypeptide. Expression of the phosphonoacetaldehyde hydrolase coding sequence in Escherichia coli under control of the E. coli tac promoter resulted in the production of enzymatically active protein with an affinity constant similar to that of the phosphonoacetaldehyde hydrolase purified from P. aeruginosa A237. This is the first nucleic sequence report of the phosphonoacetaldehyde hydrolase, an enzyme involved in the carbon–phosphorus bond cleavage.

Phosphoenolpyruvate mutase catalysis of phosphoryl transfer in phosphoenolpyruvate: kinetics and mechanism of phosphorus-carbon bond formation.

Phosphoenolpyruvate phosphomutase (PEP mutase) from Tetrahymena pyriformis catalyzes the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr). A spectrophotometric P-pyr assay consisting of the coupled actions of P-pyr decarboxylase, phosphonoacetaldehyde hydrolase, and alcohol dehydrogenase was devised to monitor mutase catalysis. The reaction constants determined for PEP mutase catalyzed conversion of PEP to P-pyr at pH 7.5 and 25 degrees C in the presence of Mg(II) are kcat = 5 s(-1), Km = 0.77 +/- 0.05 mM, and Keq = (2-9) x 10(-4). In the PEP forming direction, kcat = 100 s(-1) and Km = 3.5 +/- 0.1 microM. Retention of stereochemistry at phosphorus and strong inhibition displayed by the pyruvyl enolate analog, oxalate, have been cited as two lines of evidence that PEP mutase catalysis proceeds via a phosphoenzyme-pyruvyl enolate intermediate [Seidel, H. M., & Knowles, J. R. (1994) Biochemistry 33, 5641-5646]. In this study, single turnover reactions of oxalyl phosphate with the PEP mutase were carried out to test the formation of the phosphoenzyme intermediate. If formed. the phosphoenzyme-oxalate complex should be sufficiently stable to isolate. Reaction of the mutase with [32P]oxalyl phosphate in the presence of Mg(II)/Mn(II) cofactor failed to produce a detectable level of the [32P]phosphoenzyme-oxalate complex. In contrast, the same reaction carried out with pyruvate phosphate dikinase (PPDK), an enzyme known to catalyze the phosphorylation of its active site histidine with PEP, occurred at a rate of 4 x 10(-4) s(-1) (15% E-P formed) in the presence Mg(II) and at a rate of 3 x 10(-3) s(-1) (60% E-P formed) in the presence of Mn(II). Both oxalyl phosphate (Ki = 180 +/- 10 microM) and oxalate (Ki = 32 +/- 1O microM) were competitive inhibitors of PEP mutase catalysis, but neither displayed slow, tight binding inhibition. These results do not support the intermediacy of a phosphoenzyme-pyruvyl enolate complex in PEP mutase catalysis.

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

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStereochemical probe for the mechanism of phosphorus-carbon bond cleavage catalyzed by the Bacillus cereus phosphonoacetaldehyde hydrolaseSheng Lian Lee, Timothy W. Hepburn, William H. Swartz, Herman L. Ammon, Patrick S. Mariano, and Debra Dunaway-MarianoCite this: J. Am. Chem. Soc. 1992, 114, 19, 7346–7354Publication Date (Print):September 1, 1992Publication History Published online1 May 2002Published inissue 1 September 1992https://doi.org/10.1021/ja00045a003RIGHTS & PERMISSIONSArticle Views326Altmetric-Citations25LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Investigation of the substrate binding and catalytic groups of the PC bond cleaving enzyme, phosphonoacetaldehyde hydrolase

Kinetic studies with substrate analogs and group-directed chemical modification agents were carried out for the purpose of identifying the enzyme-substrate interactions required for phosphonoacetaldehyde (P-Ald) binding and catalyzed hydrolysis by P-Ald hydrolase (phosphonatase). Malonic semialdehyde ( K i = 1.6 mM), phosphonoacetate ( K i = 10 mM), phosphonoethanol ( K i = 10 mM), and fluorophosphate ( K i = 20 mM) were found to be competitive inhibitors of the enzyme but not substrates. Thiophosphonoacetaldehyde and acetonyl phosphonate underwent phosphonatase-catalyzed hydrolysis but at 20-fold and 140-fold slower rates, respectively, than did P-Ald. In the presence of NaBH 4, acetonylphosphonate inactivated phosphonatase at a rate exceeding that of its turnover. Sequence analysis of the radiolabeled tryptic peptide generated from [3- 3H]acetonylphosphonate/NaBH 4-treated phosphonatase revealed that Schiff base formation had occurred with the catalytic lysine. From the V m K m and V m pH profiles for phosphonatase-catalyzed P-Ald hydrolysis, an optimal pH range of 6–8 was defined for substrate binding and catalysis. The pH dependence of inactivation by acetylation of the active site lysine with acetic anhydride and 2,4-dinitrophenyl acetate evidenced protonation of the active site lysine residue as the cause for activity loss below pH 6. The pH dependence of inactivation of an active site cysteine residue with methyl methanethiolsulfonate indicated that deprotonation of this residue may be the cause for the loss of enzyme activity above pH 8.