Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)- N-Boc-3-Hydroxypiperidine

Abstract

The recombinant carbonyl reductase from Rhodococcus erythropolis WZ010 (ReCR) demonstrated strict (S)-stereoselectivity and catalyzed the irreversible reduction of N-Boc-3-piperidone (NBPO) to (S)-N-Boc-3-hydroxypiperidine [(S)-NBHP], a key chiral intermediate in the synthesis of ibrutinib. The NAD(H)-specific enzyme was active within broad ranges of pH and temperature and had remarkable activity in the presence of higher concentration of organic solvents. The amino acid residue at position 54 was critical for the activity and the substitution of Tyr54 to Phe significantly enhanced the catalytic efficiency of ReCR. The k_cat/K_m values of ReCR Y54F for NBPO, (R/S)-2-octanol, and 2-propanol were 49.17 s^-1 mM^-1, 56.56 s^-1 mM^-1, and 20.69 s^-1 mM^-1, respectively. In addition, the (S)-NBHP yield was as high as 95.92% when whole cells of E. coli overexpressing ReCR variant Y54F catalyzed the asymmetric reduction of 1.5 M NBPO for 12 h in the aqueous/(R/S)-2-octanol biphasic system, demonstrating the great potential of ReCR variant Y54F for practical applications.

Keywords: (S)-N-Boc-3-hydroxypiperidine; Rhodococcus erythropolis; asymmetric reduction; carbonyl reductase; rational design.

Scheme 1

Asymmetric bioreduction of N-Boc-3-piperidone (NBPO) using (R/S)-2-octanol or 2-propanol as co-substrate for NADH regeneration.

Figure 1

SDS-PAGE (12.5%) analysis of the purified recombinant ReCR. Lane 1, 2 μg purified ReCR with C-terminal His-tag; lane M, molecular weight marker. Coomassie Brilliant Blue R-250 was used to visualize the protein bands in the SDS-PAGE gel.

Figure 2

Structure-related sequence alignment between ReCR and its homologous proteins. 2XAA, PDB code of alcohol dehydrogenase from Rhodococcus ruber DSM 44541; PAR, alcohol dehydrogenase from Rhodococcus sp. ST-10 (GenBank accession No.: AB020760.3); ReADH, alcohol dehydrogenase from R. erythropolis DSM 43297 (GenBank accession No.: AY161280.1). The amino acid sequences of both PAR and ReADH are identical. Shown above the alignments are elements of the secondary structure of 2XAA. The numbering shown is from 2XAA. Red stars, putative catalytic residues; blue stars, residues for the coordination of structural zinc. Strictly conserved residues are highlighted with red boxes.

Figure 3

Effect of pH (A) and temperature (B) on the activity of recombinant ReCR. The relative activities of 100% represent 85.8 U/mg for NBPO reduction (solid symbols) and 88.3 U/mg for (R/S)-2-octanol oxidation (open symbols). The buffers 2-(N-morpholino)ethanesulfonic acid (MES, ■), piperazine-1,4-bisethanesulfonic acid(PIPES, ●), Tris-HCl (▲), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO, ◆) were used for the reduction reaction, while the buffers Tris-HCl (△), CAPSO (◇), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS, ▽) were used for the oxidation reaction.

Figure 4

The stability of ReCR against heat (A) and organic solvents (B). Symbols: (■) for 60 °C, (●) for 55 °C, (▲) for 35 °C. The relative activity of 100% represents 85.8 U/mg for NBPO reduction. The enzyme was incubated with organic solvent (40% (v/v) (R/S)-2-octanol, 40% (v/v) 2-octanone, 20% (v/v) 2-propanol, or 20% (v/v) acetone) at 35 °C for 3.5 h prior to the stability test against organic solvent.

Figure 5

Protein-ligand structures of ReCR with NBPO (A) and ReCR Y54F with NBPO (B). ReCR and ReCR Y54F are represented in cartoon format. Tyr54, Phe54, NADH, and NBPO are highlighted in sticks. The zinc ion is shown as a magenta sphere.