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Microbiome: When Form Doesn't Equal Function

SBKB [doi:10.1038/sbkb.2014.229]
Featured Article - November 2014
Short description: The characterization of three proteins from the human microbiome extends our understanding of PLP-dependent enzymes.

The JCSG high-throughput structural biology pipeline was used to determine the X-ray structures of three novel PLPDEs from bacteria found in the human gut and oral cavity (A). Structural analysis with bound PLP (B), together with comparison to proteins with similar active site motifs (C) and molecular simulations (D) led to the discovery of the natural substrates for these PLPDEs. Figure courtesy of Ian Wilson.

The functional annotation of enzymes remains an outstanding challenge in the post-genomic era. This challenge is particularly relevant to our understanding of the human microbiome, in light of the exploding metagenomic information opening up uncharted biological territory. The discovery of unique biochemical activities in the microbiome may offer insight into the basis for divergent host interactions with seemingly similar species.

Pyridoxal-5′-phosphate (PLP)-dependent enzymes are estimated to be involved in 4% of all catalytic reactions, and the sequence motifs involved in binding the PLP cofactor enable annotation of these enzymes as a class. However, the large number of known PLP-dependent reactions, coupled with the substantial promiscuity of individual enzymes, has frustrated their biochemical assignments.

To assist in this endeavor, Wilson, Toney and colleagues (PSI JCSG) now report the structural and biochemical characterization of three PLP-dependent enzymes—EUBREC_0560, EUBREC_2651 and PG_1327—from two members of the human microbiome, nonpathogenic Eubacterium rectale and periodontal-disease associated Porphyromonas gingivalis. The three enzymes, with structures solved to 1.7–2.1-Å resolution, adopt the type I fold associated with eight subclasses of PLP-dependent enzymes.

In their assessment of the EUBREC_0560 structure (PDB 3ELE), the authors found that the enzyme was similar to aspartate, tyrosine and alanine aminotransferases. A qualitative activity test showed that EUBREC_0560 could deaminate thirteen of the twenty amino acids, though steady-state kinetics suggested tryptophan was the natural enzyme ligand. This specificity could be rationalized using molecular dynamics simulations by the presence of neighboring hydrophobic and aromatic residues, as well as an arginine, notably mobile in the apo crystal structure, which the authors postulate forms a cation-π interaction with the substrate.

Though structure similarity matching for PG_1327 (PDB 3G0T) suggested that this enzyme would be specific for charged substrates, detailed kinetic parameters instead pointed to aromatic amino acids as the natural ligands, with decreased activity on acidic substrates.

Structural comparisons of EUBREC_2651 (PDB 3F0H) directed the authors to smaller substrates, though the enzyme was active on a large range of amino acids. The unusual reactivity seen with phosphoserine suggested that the enzyme could be involved in serine biosynthesis, but some unexpected outcomes in biochemical assays raised questions about this assignment. The authors note that the genetic context of EUBREC_2651 is linked to teichoic acid biosynthesis; future work will be needed to unequivocally identify the function of this intriguing enzyme.

Catherine Goodman

References

  1. N.M. Fleischman et al. Molecular characterization of novel pyridoxal-5′-phosphate-dependent enzymes from the human microbiome.
    Protein Sci. 23, 1060-76 (2014). doi:10.1002/pro.2493

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Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health