Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa

Suyan Liu; T L Yahr; D W Frank; J T Barbieri

doi:10.1128/jb.179.5.1609-1613.1997

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Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa

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Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa

Suyan Liu, T L Yahr, D W Frank and J T Barbieri

Journal of bacteriology, Vol.179(5), pp.1609-1613

03/1997

DOI: 10.1128/jb.179.5.1609-1613.1997

PMCID: PMC178873

PMID: 9045820

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Abstract

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.

Amino Acid Sequence

Poly(ADP-ribose) Polymerases - immunology

Recombinant Fusion Proteins - isolation & purification

Immunodominant Epitopes - analysis

Molecular Weight

Mutagenesis, Site-Directed

NAD+ Nucleosidase - metabolism

Trypsin Inhibitor, Kunitz Soybean - metabolism

Molecular Sequence Data

ras Proteins - metabolism

Recombinant Fusion Proteins - metabolism

Poly(ADP-ribose) Polymerases - metabolism

Poly(ADP-ribose) Polymerases - genetics

Bacterial Toxins

Pseudomonas aeruginosa - enzymology

ADP Ribose Transferases

Kinetics

Poly(ADP-ribose) Polymerases - chemistry

Binding Sites

NAD - metabolism

Details

Title: Subtitle: Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa
Creators: Suyan Liu - Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA
T L Yahr
D W Frank
J T Barbieri
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.179(5), pp.1609-1613
DOI: 10.1128/jb.179.5.1609-1613.1997
PMID: 9045820
PMCID: PMC178873
NLM abbreviation: J Bacteriol
ISSN: 0021-9193
eISSN: 1098-5530
Publisher: United States
Grant note: R01-AI-30162 / NIAID NIH HHS R01-AI-31665 / NIAID NIH HHS R04-AI-01087 / NIAID NIH HHS
Language: English
Date published: 03/1997
Academic Unit: Microbiology and Immunology
Record Identifier: 9984002390202771

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