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Journal of Bacteriology, July 2005, p. 4573-4583, Vol. 187, No. 13
0021-9193/05/$08.00+0     doi:10.1128/JB.187.13.4573-4583.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Epimerase Active Domain of Pseudomonas aeruginosa AlgG, a Protein That Contains a Right-Handed ß-Helix

Stephanie A. Douthit,1,4 Mensur Dlakic,1 Dennis E. Ohman,2,3 and Michael J. Franklin1,4*

Department of Microbiology,1 Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717,4 Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia,2 McGuire Veterans Affairs Medical Center, Richmond, Virginia3

Received 21 November 2004/ Accepted 30 March 2005

The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of ß1-4-linked O-acetylated D-mannuronate (M) and L-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed ß-helix (RHßH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHßH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHßH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHßH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHßH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second D-mannuronate residue to L-guluronate along the epimerase catalytic face.

* Corresponding author. Mailing address: Department of Microbiology, 109 Lewis Hall, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2420. Fax: (406) 994-4926. E-mail: umbfm{at}montana.edu.

Journal of Bacteriology, July 2005, p. 4573-4583, Vol. 187, No. 13
0021-9193/05/$08.00+0     doi:10.1128/JB.187.13.4573-4583.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Aachmann, F. L., Svanem, B. I. G., Guntert, P., Petersen, S. B., Valla, S., Wimmer, R. (2006). NMR Structure of the R-module: A PARALLEL beta-ROLL SUBUNIT FROM AN AZOTOBACTER VINELANDII MANNURONAN C-5 EPIMERASE. J. Biol. Chem. 281: 7350-7356 [Abstract] [Full Text]  


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