An Assay for Measuring the Activity of <em>Escherichia coli</em> Inducible Lysine Decarboxyase

Abstract

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5-9) and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1-2. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB, the arginine decarboxylase AdiA, the lysine decarboxylase LdcI and the ornithine decarboxylase SpeF. All of these enzymes utilize pyridoxal-5'-phosphate as a co-factor and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 Å, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp). The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth. We have demonstrated that ppGpp is a specific inhibitor of LdcI. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al., that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the α-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane). L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N'-bistrinitrophenyllysine (TNP-lysine), respectively. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not. The linear range of the assay was determined empirically using purified cadaverine.