Functional annotation and kinetic characterization of PhnO from Salmonella enterica - PubMed (original) (raw)

Functional annotation and kinetic characterization of PhnO from Salmonella enterica

James C Errey et al. Biochemistry. 2006.

Abstract

Phosphorus is an essential nutrient for all living organisms. Under conditions of inorganic phosphate starvation, genes from the Pho regulon are induced, allowing microorganisms to use phosphonates as a source of phosphorus. The phnO gene was previously annotated as a transcriptional regulator of unknown function due to sequence homology with members of the GCN5-related N-acyltransferase family (GNAT). PhnO can now be functionally annotated as an aminoalkylphosphonic acid N-acetyltransferase which is able to acetylate a range of aminoalkylphosphonic acids. Studies revealed that PhnO proceeds via an ordered, sequential kinetic mechanism with AcCoA binding first followed by aminoalkylphosphonate. Attack by the amine on the thioester of AcCoA generates the tetrahedral intermediate that collapses to generate the products. The enzyme also requires a divalent metal ion for activity, which is the first example of this requirement for a GNAT family member.

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Figures

Figure 1

Figure 1

Double reciprocal plot of initial rate data at varying _S_-1AEP concentrations and fixed concentrations of acetyl-CoA at 10 μM (♀), 20 μM (○), 30 μM (formula image), 60 μM (→) and 250 μM (†). The pattern of intersecting lines is indicative of a sequential kinetic mechanism.

Figure 2

Figure 2

Dependence of _k_cat and _k_cat/K _S_-1AEP on pH at saturating levels of acetyl-CoA and Ni2+. The dotted and solid lines are fits to Eqs 7 and 8, respectively. The _k_cat pH profile revealed the presence a single ionizable group with a pK value of 8.2 (± 0.1). The _k_cat/K _S_-1AEP profile was fit to Eq. 8, which describes the dependence on 2 groups with similar pK values of 7.5 (± 0.1) and Eq. 7, which describes the dependence on 1 group (dotted line).

Figure 3

Figure 3

Reciprocal plots of the solvent kinetic isotope effect determined for PhnO using acetyl-CoA (top panel) or _S_-1AEP (bottom panel) as the variable substrate, in the presence of saturating concentrations of Ni2+. The symbols are the experimentally determined values in H2O (♀)or 90% D2O (formula image), while the lines are fits of the data to Eq. 9.)

Scheme 1

Scheme 1

Pathways for phosphonate catabolism. (I) The C-P lyase pathway acts on alkylphosphonates as well as aminoalkylphosphonates. (II) The phosphonatase pathway that uses 2-aminoethylphosphonate and cleaves the C-P bond in a two-step transamination and hydrolysis process. (III) The S. enterica phn gene locus; phnR encodes a putative aminoalkylphosphonate transport repressor, phnS-V encode putative aminoalkylphosphonate transport components, phnW encodes a 2-aminoethylphosphonate transaminase, phnX encodes a 2-phosphonoacetaldehyde hydrolase, phnA encodes a putative phosphonoacetate hydrolase, phnB encodes a putative phosphonoacetate transporter, phnO encodes an aminoalkylphosphonic _N_-acetyltransferase.

Scheme 2

Scheme 2

Proposed kinetic and chemical mechanisms for aminophosphonate _N_-acetylation by PhnO

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