Broad Substrate Specificity and High Catalytic Activity of Sphingomonadaceae PhoK-Type Phosphatases Implicated in Flame-Retardant Degradation

Abstract

The first two enzymes recognized to be PhoK-type phosphatases were from Sphingobium sp. TCM1 (Sb-PhoK) and Sphingomonas sp. BASR1 (Sm-PhoK) which were utilized in bioremediation of organophosphate flame-retardants and heavy metal contamination, respectively. The PhoK-type phosphatases are members of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of diesterases that have evolved a phosphatase activity. These enzymes were noted for very high activity with model compounds compared to other alkaline phosphatases, but very little was known about their substrate specificity or the activity with phosphomonoesters derived from flame-retardants or other environmental phosphoesters. Bioinformatics analysis has been utilized to identify PhoK homologues from a large group of Sphingomonadaceae family members including additional species that are known or suspected to utilize the organophosphate flame-retardants as nutrient sources. Nine homologues were selected for kinetic characterization using a synthesized library of organophosphate monoesters derived from flame-retardants, environmental phosphoesters, and biological monophosphates. The Sphingomonadaceae PhoK enzymes were found to have high enzymatic efficiency against a broad range of substrates. Against phenyl phosphate Sm-PhoK has a k _cat of 1100 s^-1 and a k _cat/K _m of 1.8 × 10⁶ M^-1 s^-1. The best overall activity was observed with the homologue from Sphingobium yanoikuyae (Sy-PhoK), another species known to degrade organophosphate flame-retardants. This enzyme hydrolyzed all tested substrates with an efficiency greater than 3 × 10⁴ M^-1 s^-1. The high catalytic activity and remarkably broad substrate specificity make the Sphingomonadaceae PhoK enzymes particularly suited for bioremediation as well as commercial applications where high turnover will be advantageous.

1

(a) Metabolic pathway for organophosphate flame-retardant degradation evolved in Sphingobium sp. TCM1. (b) Phosphomonoesters tested in this work.

2

Sequence similarity network of PhoK-homologues shown with an alignment score cutoff of 175. Sequences primarily cluster according to genus with the members of the Sphingomonadaceae family (Sphingopyxis (dark blue), Sphingomonas (yellow), Sphingobium (green), and Novosphingobium (red) clustering together). Other clusters are identified by the primary genus found in the cluster. The characterized protein Sm-SRS2-PhoK not found in the Sphingomonadaceae cluster is labeled. Singlets and clusters with less than 4 sequences have been omitted for clarity.

3

Hydrolysis of 2.5 mM 1,3-dichloroisopropyl phosphate (4) by 4.1 nM Sb-PhoK followed by ³¹P NMR. Panel (a) shows the ³¹P NMR spectra 1,3-dichloroisopropyl phosphate (4) with resonance at 2.78 ppm before addition of enzyme. Panel (b) shows appearance of the phosphate product at 2.01 ppm 70 min after addition of enzyme. Panel (c) shows reaction after 235 min, and panel (d) shows product after 460 min. Panel (e) shows exponential curve fit to NMR data.