Structural Insights Into Novel Microbial Metalloenzymes

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van Staalduinen, Laura
Biochemistry , Protein Crystallography , Structural Biology
Metalloproteins represent a large portion of the total proteome. When bound to a protein a metal ion influences both protein stability and function through structural, catalytic or regulatory roles. Discovery of a metal ion cofactor presents new insight into both the structural and functional aspects of a protein and can be essential for the elucidation of the functional and mechanistic details of a protein of interest. The cupin, 2-oxoglutarate/Fe2+-dependent oxygenases (2OG oxygenases) and the di-iron oxygenase families of metalloproteins exemplify the diversity and catalytic potential of a metal ion cofactor, as well as the conservation of 3-dimensional fold and structural features in proteins with similar functions. The structural and biochemical characterization of three novel microbial metalloenzymes are presented; two Escherichia coli hypothetical proteins of previously unknown function, E. coli cupin sugar isomerase (EcSI) and a 2OG oxygenase, YcfD, and the novel microbial carbon-phosphorus (C-P) bond cleavage enzyme, PhnZ, are presented. In each case the identification of a metal ion cofactor and structure determination led to important functional insights. EcSI is encoded by a gene that is highly conserved among pathogenic bacteria. It has been identified as a sugar isomerase with specificity for the rare sugar D-lyxose as well as D-mannose based on structural homology to the cupin phosphoglucose isomerases, suggesting a role for EcSI in metabolism of alternative carbon sources. Structural homology of YcfD, the second metalloenzyme, to the 2OG oxygenase family, particularily human proteins involved in ribosome assembly, combined with evidence that YcfD interacts with the essential ribosomal protein L-16 provides the first evidence of translational regulation by a 2OG oxygenase in E. coli. The third metalloenzyme, PhnZ, was previously identified as an iron dependent oxygenase. Structural characterization revealed that PhnZ possesses a di-iron cofactor and shows significant structural homology to a di-iron oxygenase, providing structural evidence for its novel mechanism of C-P bond cleavage. Combined, these three structures also highlight several features of metal ion-enzyme interaction and regulation mechanisms employed by metalloenzymes as well as the importance of structure in the elucidation of functional and mechanistic characteristics of a protein.
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