Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

Abstract

Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide called alginate during infection in the lungs of cystic fibrosis patients. A mutation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a nonmucoid (nonalginate-producing) phenotype and showed reduced activities of succinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis. We have cloned and characterized the sucCD operon encoding the ␣ and ␤ subunits of Scs from P. aeruginosa and have studied the role of Scs in generating GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor and, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor. In the presence of inorganic orthophosphate, succinyl-CoA, and an equimolar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount of ATP and GTP. Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macromolecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is also shown to exhibit a broad NDP kinase activity. In the presence of inorganic orthophosphate (P i ), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP. Scs was previously shown to copurify with Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is also known to exist in two forms, a 16-kDa cytoplasmic form predominant in the log phase and a 12-kDa membrane-associated form predominant in the stationary phase. We have observed that the 16-kDa Ndk-Scs complex present in nonmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifically present in mucoid cell predominantly synthesizes GTP and UTP but not CTP. Such regulation may promote GTP synthesis in the stationary phase when the bulk of alginate is synthesized by mucoid P. aeruginosa.Our laboratory is interested in studying the regulation of biosynthesis of alginate in Pseudomonas aeruginosa, an opportunistic pathogen. A number of cystic fibrosis (CF) isolates have been studied for their enhanced synthesis and secretion of alginate (16). Alginate is a polymer of D-manuronic and its C-5 epimer L-guluronic acid. GDP-mannuronic acid, derived from GDP-mannose, is the building block of alginate, which requires mannose 1-phosphate and GTP as precursors (17). Thus, alginate synthesis demands a steady supply of GTP for every molecule of mannuronic acid incorporated into a molecule of alginate. Recently, Kamath et al. (9) have shown that the mucoid strains of P. aeruginosa retain an active form of elastase in their periplasm, and this periplasmic elastase acts on 16-kDa Ndk to generate a truncated 12-kDa form that is membrane associated. It has been demonstrated that the membrane-associated 12-kDa nucleoside diphosphate (NDP) kinase (Ndk), in complex with pyruvat...

Characterization of the last step of the aerobic phenylacetic acid degradation pathway

“…By sequencing the chromosomal regions flanking the mini-Tn5araC-P BAD insertion sites, we realized that one of the mutants, the P. putida KT2442-150A strain (Table 1, Fig. 2A), did not contain the transposon insertion within the paa gene cluster (formerly pha cluster) (Jiménez et al, 2002(Jiménez et al, , 2004) but rather on a gene (TIGR locus name PP4185 of the annotated P. putida genome; http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database =gpp) whose putative product showed 97 and 88 % amino acid sequence identity with the sucD gene products of P. aeruginosa (Kapatral et al, 2000) and E. coli (Buck et al, 1986), respectively. The sucD gene encodes the a subunit of the succinyl-CoA synthetase (SucCD) that converts succinyl-CoA into succinate in the TCA cycle (Fig.…”

Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7 T during Degradation of 3,3′-Dithiodipropionic Acid

“…Due to the possibility of substituting the cofactor Mg 2ϩ by Mn 2ϩ and the coenzyme ATP by GTP, SucCD Am shows behavior similar to that of other SucCDs from Gram-negative bacteria (21).…”

“…In Pseudomonas aeruginosa SucCD has a very broad nucleotide specificity and is able to use ADP, GDP, UDP, and CDP in combination with inorganic phosphate and succinyl-CoA to synthesize the corresponding nucleoside triphosphates (21). In eukaryotes, SucCDs with higher specificity are found.…”

Functional Genomics of Stress Response in Pseudomonas putida KT2440

“…PcaIJ catalyzes the penultimate step in the degradation of benzoate and protocatechuate, the transfer of coenzyme (CoA) to ␤-ketoadipate (49,58,63,89). The succinyl-CoA synthetase is a key enzyme of the tricarboxylic acid cycle and converts the product of the final step of the ␤-ketoadipate pathway, succinyl-CoA, in a three-step reaction into succinate and nucleoside triphosphate (60). Complementation of the mutants in trans restored the initial growth rate of the wild type.…”