Microbial hydroxylases were screened for the capacity to effect direct hydroxylation of proline and pipecolinic acid, based on genomic information. Of the eight candidates screened, 2-oxoglutarate-dependent hydroxylase from Streptosporangium roseum NBRC 3776T and aspartyl/asparaginyl β-hydroxylase from Catenulispora acidiphila NBRC 102108T showed both proline and pipecolinic acid hydroxylation activities. In the case of l-proline hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-l-proline and cis-4-hydroxy-l-proline, and cis-4-hydroxy-l-proline was preferentially produced. In the case of l-pipecolinic acid hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-l-pipecolinic acid and cis-5-hydroxy-l-pipecolinic acid. While the former enzyme preferentially produced cis-3-hydroxy-l-pipecolinic acid, the latter enzyme preferentially produced cis-5-hydroxy-l-pipecolinic acid.
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