Structural and functional characterization of PaaK1 & PaaK2: phenylacetate CoA ligase paralogs from Burkholderia cenocepacia J2315.




Law, Adrienne

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Aromatic compounds comprise approximately 25% of the Earth`s biomass. Accordingly, turnover of these thermostable compounds is essential to the biogeochemical carbon cycle. Specialized microbial enzymatic pathways are largely responsible for mineralization of aromatic compounds, and are extensively studied for bioremediation purposes. The phenylacetic acid degradation pathway (PAA) is of particular interest as it is utilized for degradation of a multitude of aromatic compounds, including environmental pollutants, and appears to be widely distributed among bacteria. Intriguingly, the PAA pathway has also been implicated as a virulence factor in the cystic fibrosis pathogen, Burkholderia cenocepacia. As such, detailed biochemical characterization of the PAA pathway holds great potential for improving bioremediation strategies for aromatic pollutants, as well as understanding carbon source utilization during B. cenocepacia infection. A striking feature of the PAA pathway in B. cenocepacia is the presence of two genes encoding the phenylacetate CoA ligase (PCL) enzyme (paaK1 and paaK2), responsible for the initial CoA activation of phenylacetic acid. PCLs are members of the adenylate forming enzyme superfamily. However, sequence alignments reveal several intriguing features, including a potentially novel microdomain consisting of the initial ~80 N-terminal residues, which possesses percent identity to any structurally characterized family member. Furthermore, this superfamily utilizes a complicated catalytic mechanism, exploiting several conserved motifs during the reaction process. The precise roles of many key conserved residues are not yet well understood, especially during the pre-adenylation, ATP bound state, for which few high quality crystal structures exist. In order to define the early stages of the catalytic mechanism, and to assess how the divergent polypeptide region may impact the PaaK enzymes, we have pursued a detailed structural characterization of the paralogs, complemented with functional assessments. Specifically, we have produced a 1.6 Å resolution crystal structure of PaaK1 in complex with ATP, which reveals a novel helical bundle arrangement at the N-terminal domain never before seen in this superfamily. Remarkably, homodimerization of PaaK1 appears to reconstitute potentially important β sheet interactions observed in the classical N-terminal arrangement of family members. Moreover, our structure is one of few which contain well ordered β and γ phosphates, allowing for detailed examination of significant protein-ATP interactions with conserved catalytic residues. To better comprehend the roles of these residues over the course of the reaction, we have produced additional crystal structures of PaaK1 and PaaK2 in complex with the phenylacetyl adenylate intermediate. Notably, PaaK2 was captured following the domain reorientation, poised to catalyze thioesterification of phenylacetyl adenylate, providing insight into the later stages of the reaction process. Furthermore, the intermediate co-structures divulge the location of the aryl substrate binding pocket for both paralogs. Detailed comparisons of the binding pockets accompanied kinetic characterizations for both paralogs, demonstrating that PaaK2 possesses an apparent KM of 150 μM for phenylacetic acid, more than double that of PaaK1 (62 μM). Our findings provide preliminary evidence for distinct functional roles of the PaaK paralogs in B. cenocepacia, while imparting additional insight into catalytic roles of conserved residues within the adenylate forming superfamily at large.



aromatic compounds, phenylacetic acid