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 × 10(6) 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 × 10(4) 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.