Determination of <i>Francisella tularensis</i> AcpB Acid Phosphatase Substrate Preferences

Abstract

The Francisella speciesencode 4 main acid phosphatases (Acp) that are potentially involved in pathogenesis through currently unknown mechanisms. Only 2 of these enzymes, AcpA and AcpC, have been biochemically characterized to date. In this work we describe the catalytic properties of Francisella tularensis AcpB utilizing an array of 120 phosphorylated substrates. In contrast to most acid phosphatases, the purified enzyme showed a narrow range of substrate preferences, with the highest affinity towards thiamine phosphate (Km = 150 µM). Francisella species do not possess a thiamine biosynthetic pathway even though vitamin B1 is indispensable in numerous cellular functions. Consequently, thiamine should be incorporated from the environment, in this case, from the host cell. Our results suggested that AcpB could provide the hydrolytic activity necessary to transform the nontransportable phosphorylated vitamin B1 present in tissues to a form that can be absorbed by the intracellular pathogen.

Functional characterization of LotP from Liberibacter asiaticus

“…In a subsequent enzymatic assay, LotP's activity towards several nucleosides was tested in parallel. Malachite green was used to detect the P i released by the enzyme action (Crowe et al ., ). The results demonstrated that LotP preferentially used ATP as an enzyme substrate (Fig.…”

“…B. Steady‐state kinetic characterization of ATP ase activity. Initial rates of the reactions were determined at fixed concentrations of ATP , and the P i released was quantified by using Malachite green reagent as previously described (Crowe et al ., ).…”

“…Phosphate released after enzymatic ATP hydrolysis was quantified using the malachite green assay in order to assess ATPase activity (Crowe et al ., ). Protease activity was assessed by using azocasein as the substrate in the presence and absence of ATP and using proteinase K as a positive control (Secades and Guijarro, ).…”