Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli

Abstract

The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes (A-D). Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes ( btuC, btuE, btuD, btuF, and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and co-expressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS methylases that suffer from poor solubility during overproduction. This strategy markedly enhances the uptake of cobalamin into the cytoplasm and improves the solubility of the target enzymes significantly.

Scheme 1

Steps involved in the synthesis of HEP-CMP.

Scheme 2

Steps involved in the synthesis of HPP-CMP.

Figure 1

Various natural products that contain methyl moieties installed by cobalamin-dependent RS methylases. Compounds with structures shown represent products of class B RS methylases that have been studied in vitro. One arrow indicates the actual product from an in vitro reaction, while two arrows indicate the final natural product whose biosynthesis involves a class B RS methylation step.

Figure 2

Structures of (a) hydroxocobalamin, (b) adenosylcobalamin, (c) methylcobalamin, and (d) cobinamide.

Figure 3. Cobalamin uptake system in E. coli.

Cobalamin is transported across the outer membrane by the TonB-dependent outer membrane protein BtuB. BtuF, a periplasmic membrane transporter, then delivers the cobalamin to BtuC and BtuD, which subsequently transport cobalamin across the inner membrane into the cytosol.

Figure 4

The pBAD42-BtuCEDFB vector. (a) A plasmid map of the vector. Expression of btuCEDFB is driven by an arabinose inducible araBAD promoter. (b) The cloning regions of the btuCEDFB operon. The optimized ribosome binding sites are shown in red, while the start (bold) and stop (underlined) codons are also displayed.

Figure 5

Relative concentrations of MeCbl (orange), OHCbl (blue), and AdoCbl (red) in E. coli cultured in LB, M9, and M9-ethanolamine (Eth) media with and without pBAD42-BtuCEDFB normalized to the amount of AdoCbl in E. coli cultured in M9-ethanolamine media with the pBAD42-BtuCEDFB plasmid.

Figure 6

LC-MS/MS analysis of the cobalamin content of (a) as-isolated Fom3 (20 μM) and (b) as-isolated TsrM. (a) As-isolated Fom3 contains 18% OHCbl (blue line), 0% AdoCbl (black line) and 82% MeCbl (red line). (b) TsrM contains 61% OHCbl (blue line), 39% AdoCbl (black line) and <1% MeCbl (red line).

Figure 7

Time-dependent formation of HPP-CMP (black), SAH (red), and 5′-dA (blue) by Fom3 (9 μM) in the presence of 1.5 mM HEP-CMP, 1.5 mM SAM, 1 mM methyl viologen and 2 mM NADPH.

Figure 8

(a) UV-Vis spectrum of PhpK (6.5 μM) treated with potassium cyanide. (b) LC-MS/MS analysis of as-isolated PhpK, which contains 8% AdoCbl (black), 15% OHCbl (blue), and 77% MeCbl (red).

Figure 9

(a) LC-MS/MS analysis of TsrM overexpressed in the ΔbtuR strain of E. coli BW25113 reveals no bound AdoCbl (black). (b) Time-dependent formation of MeTrp by TsrM (0.1 μM) in the presence of SAM (1 mM) and Trp (1 mM).