To date, there are no examples of enzymes that catalyze the hydroxylation of 3-carboxylated piperidine (nipecotic acid). To address this gap, a select pool of enzymes were screened for reaction on nipecotic acid (Fig. and Table in the Supporting Information). trans-P4H and P4H810 under the above-described optimized conditions and two P450BM3 variants, which were reported to induce hydroxylation of N-protected heterocycles, were examined, but exhibited no hydroxylation activities against (S)-nipecotic acid (L-Nip), (R)-nipecotic acid (D-Nip), and their protected derivatives. In light of the structural resemblance of D-Nip (10) to ectoine and their similar charge distributions, ectoine 5-hydroxylase from Sphingopyxis alaskensis (SaEctD), an ectoine hydroxylase that has been identified to convert ectoine to 5-hydroxyectoine in its biosynthetic pathway, was next tested for the reaction. Initial attempt resulted in the production of trans-4-hydroxy-D-Nip with 90% conversion, whose structure was confirmed by COSY NMR analysis and X-ray crystallography of a dinitrobenzamide derivative. (see Supporting Information for detail). Increasing amount of a-KG from 2.5 eq. to 5.0 eq. improved the conversion to 100%, which was also amenable to use at higher substrate concentration (15 mM). The Michaelis-Menten kinetic parameters of EctD toward D-Nip were analyzed, and kcat and KM were determined to be