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

Abstract

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...

Caught in the Act: The Structure of Phosphorylated β-Phosphoglucomutase from Lactococcus lactis,

“…Superposition of the two structures reveals a conserved active-site platform within the core domain comprised of four peptide loops (10) (see Figure 5 for superposition (a) and schematic (b)). These four loops correspond to the four conserved sequence motifs within the HAD enzyme family which had been noted previously by us (10,26) and by other investigators (4,5,24,27,28). The structures of 2-haloalkanoic acid dehalogenase (4), phosphonatase (10), Ca 2+ -ATPase (29), phosphoserine phos- We are most intrigued by how the four phosphotransferases have become specialized to perform their specific catalytic function.…”

Comparative genomic, proteomic and exoproteomic analyses of three Pseudomonas strains reveals novel insights into the phosphorus scavenging capabilities of soil bacteria

“…To date, PhnD, which is located in an operon with genes encoding the promiscuous C‐P lyase (PhnF‐M), is the only characterized phosphonate transporter (Baker et al ., 1998). A number of putative SBPs responsible for phosphonate transport were identified in the Pseudomonas strains genomes (Fig.…”

“…A total of 267 proteins were significantly enriched [ t ‐test, P value ≤ 0.05, fold‐change (log2) ≥ 1.5] during Pi depletion and there was concordance between the two datasets. These enriched proteins included the transmembrane and ATP‐binding domains of the Pst system (PstC, PstA, PstB), a 2‐aminoethylphosphonate (2‐AEP)–specific phosphonatase (PhnX, PhnW) (Jiang et al ., 1995; Baker et al ., 1998; White and Metcalf, 2007), proteins involved in lipid remodelling (PlcP, DagK, OlsA, OlsB, Cfa, TauD) (Liu and Hulett, 1998; Antelmann et al ., 2000; Zavaleta‐Pastor et al ., 2010; Carini et al ., 2015; Sebastian et al ., 2016), a putative intracellular phosphatase (UxpA) and the twin‐arginine translocation (TAT) pathway (Putker et al ., 2013) (Table 2). Proteins for both starch (MalQ, GlgE, GlgX, GlpA and GlpB) and polyhydroxyalkanoic acid (PhaA, PhaG, PhaC) biosynthesis (carbon storage) were also enriched during Pi stress (Supporting Information Table S4).…”

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

“…1) [2,3]. Phosphonates are synthesized in microbes and lower animals to carry out specialized functions which include microbe eradication, cell-cell signaling, host infection and/or persistence, or phosphorus storage (reviewed in [4] and references therein).…”