Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis-Selective Bacterial Proline Hydroxylases that Discriminates l-Proline

Proline hydroxylases are iron(II)/2-oxoglutarate-dependent enzymes that hydroxylate l-proline and derivatives, such as lpipecolic acid, which is the six-membered-ring homologue of l-proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l-pipecolic acid and trans-3- and trans-4-methyl-l-proline, but virtually no l-proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l-proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l-proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site-directed mutagenesis in GetF did not yield variants that accepted l-proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l-proline relative to that of preferred substrates.