Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway

Abstract

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the β-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.

Keywords: biosynthesis; catalytic mechanisms; enzymes; lincosamides; pyridoxal 5′-phosphate.

Figure 1

LC-MS analysis of CcbF and CcbF/Ccb4 coupled reactions with 5β. Peaks correspond to [M+H]⁺ and [M+Na]⁺ signals from positive ion ESI. a) CcbF reaction with 5β and without derivatization, b) after treatment with DNPH. c) CcbF/Ccb4 coupled reaction with 5β and without derivatization, b) after treatment with DNPH.

Figure 2

LC-MS analysis of LmbF-catalyzed C–S bond cleavage of 5β. Peaks correspond to [M+H]⁺ and [M+Na]⁺ signals from positive ion ESI. a) Direct analysis without derivatization, b) after treatment with mBBr.

Figure 3

LC-MS analysis of coupled enzymatic assays using LmbF, LmbG, Ccb4. Peaks correspond to [M+H]⁺ and [M+Na]⁺ signals from positive ion ESI. a) LmbF/LmbG coupled reaction without derivatization, b) after the treatment with mBBr. c) LmbF/Ccb4 coupled reaction without derivatization, d) after treatment with mBBr. e) LmbF/LmbG/Ccb4 coupled reaction without derivatization, f) after treatment with mBBr.

Scheme 1

Structures of representative lincosamide antibiotics.

Scheme 2

Proposed biosynthetic pathways for lincosamide antibiotics.

Scheme 3

Preparation of the putative substrate of CcbF (5β). a) hydrazine, reflux, 22 h, 57%; b) 1H-imidazole-1-sulfonyl azide hydrochloride, CuSO₄, K₂CO₃, MeOH/H₂O, 35 h, 51%; c) PMBCl, NaH, TBAI, DMF, rt, 12 h, 79%; d) Br₂, DCM, 0 °C, 15 min; e) N-Cbz-L-cysteine benzyl ester, TBAHS, Na₂CO₃, AcOEt, 11 h, 38% over 2 steps; f) PMe₃, THF/H₂O, rt, 20 h; g) N-methyl-L-proline, EDC, HOBt, DMF, rt, 12 h, 26% over 2 steps; h) TFA, DCM, rt, 30 min, 31%; i) H₂, Pd(OH)₂/C, AcOH, MeOH/H₂O, rt, 4 h, 70%..

Scheme 4

Proposed mechanisms for PLP-dependent reactions catalyzed by LmbF and CcbF.