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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.