Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa.
Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53). N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity. Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain. N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity. Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53. E385 was also identified as a potential active site residue of Exo53.
Liu
S
S
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Yahr
T L
TL
Frank
D W
DW
Barbieri
J T
JT
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001665:Binding Sites; D016056:Immunodominant Epitopes / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D001427:Bacterial Toxins; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins; D009243:NAD; D014360:Trypsin Inhibitor, Kunitz Soybean; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S; D009244:NAD+ Nucleosidase; D018631:ras Proteins