Defining a Structural and Kinetic Rationale for Paralogous Copies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J2315

Adrienne Law,Martin J Boulanger

doi:10.1074/jbc.m111.219683

Abstract

The phenylacetic acid (PAA) degradation pathway is the sole aerobic route for phenylacetic acid metabolism in bacteria and facilitates degradation of environmental pollutants such as styrene and ethylbenzene. The PAA pathway also is implicated in promoting Burkholderia cenocepacia infections in cystic fibrosis patients. Intriguingly, the first enzyme in the PAA pathway is present in two copies (paaK1 and paaK2), yet each subsequent enzyme is present in only a single copy. Furthermore, sequence divergence indicates that PaaK1 and PaaK2 form a unique subgroup within the adenylate-forming enzyme (AFE) superfamily. To establish a biochemical rationale for the existence of the PaaK paralogs in B. cenocepacia, we present high resolution x-ray crystal structures of a selenomethionine derivative of PaaK1 in complex with ATP and adenylated phenylacetate intermediate complexes of PaaK1 and PaaK2 in distinct conformations. Structural analysis reveals a novel N-terminal microdomain that may serve to recruit subsequent PAA enzymes, whereas a bifunctional role is proposed for the P-loop in stabilizing the C-terminal domain in conformation 2. The potential for different kinetic profiles was suggested by a structurally divergent extension of the aryl substrate pocket in PaaK1 relative to PaaK2. Functional characterization confirmed this prediction, with PaaK1 possessing a lower K(m) for phenylacetic acid and better able to accommodate 3' and 4' substitutions on the phenyl ring. Collectively, these results offer detailed insight into the reaction mechanism of a novel subgroup of the AFE superfamily and provide a clear biochemical rationale for the presence of paralogous copies of PaaK of B. cenocepacia.

Highlights

Terization of the bioprocessing enzymes comprising such pathways offers opportunities to engineer novel bioremediation and biofuel production strategies
With PaaK1 and PaaK2 catalyzing the first and only committed step of the pathway [34], these enzymes are perfectly positioned to control the flow of phenylacetic acid into the PAA pathway thereby contributing to the nutritional requirements of B. cenocepacia in infected cystic fibrosis patients
High resolution crystal structures of PaaK1 and PaaK2 captured at different catalytic stages offer insight into the dynamic cycling of the P-loop, which likely serves to promote conformational change in the C-terminal domain, and the subtle, yet critical, repositioning of multipurpose active site residues such Arg326 and Glu241

Summary

Introduction

Terization of the bioprocessing enzymes comprising such pathways offers opportunities to engineer novel bioremediation and biofuel production strategies. Many structurally related compounds, including aromatic pollutants styrene and ethylbenze, are converted to phenylacetate prior to metabolism via the PAA pathway [4, 5]. Through high resolution x-ray co-crystal structures with both ATP and the phenylacetyl adenylate intermediate, and kinetic assays, we propose a biochemical rationale for the presence of the two isozymes and include detailed analyses of the unique structural features such as the novel N-terminal microdomain. These data offer rare insight into multifunctional roles for key active site residues and substructures and are discussed with respect to the general catalytic mechanism AFE superfamily enzymes

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Results

Conclusion