Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

Abstract

Edited by Ruma BanerjeeFlavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH 2 , oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate tryptophan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ⑀-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of L-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1 and S2 and Figs. S1-S17. The atomic coordinates and structure factors (codes 6H43, 6H44, and 6IB5) have been deposited in the Protein Data Bank (http://wwpdb.org/). . 3 The abbreviations used are: FDH, flavin-dependent halogenase; aa, amino acid(s); ESI, electrospray ionization; HSQC, heteronuclear single-quantum coherence; r.m.s.d., root mean square deviation; ROESY, rotating frame nuclear Overhauser effect spectroscopy; Bicine, N,N-bis(2-hydroxyethyl)-glycine; H-bond, hydrogen bond; Ni-NTA, nickel-nitrilotriacetic acid; RR-ADH, Rhodococcus ruber alcohol dehydrogenase; PDB, Protein Data Bank.Figure 8. Steric conflicts upon placing L-Trp from Trp-RebH in Trp-Thal and vice versa. Minor clashes are indicated as small green hexagons, and more severe clashes are shown as larger red hexagons. a, L-Trp from Trp-RebH (PDB code 2E4G) was placed in Trp-Thal. Clashes mainly occur between the L-Trp indole and the side chains of conserved residues Phe-112 and Trp-466. b, L-Trp from Trp-Thal was placed in Trp-RebH (PDB code 2E4G). Clashes mainly occur between the L-Trp indole and the side chain of Asn-470 and between the L-Trp carboxylate and the side chain...

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

“…Investigating the location and impact of mutations can support to provide a rationale for increased thermostability. The recently resolved crystal structure of Thal‐WT served to localize the mutations known in Thal‐GWV‐NT . As observed for other thermostable enzyme variants such as RebH it was not surprising that the majority of mutations is located on the protein surface remote from the active site (Figure ) .…”

“…The recently resolved crystal structure of Thal-WT served to localize the mutations known in Thal-GWV-NT. [28,43] As observed for other thermostable enzyme variants such as RebH it was not surprising that the majority of mutations is located on the protein surface remote from the active site ( Figure 3). [36] An increase of surface charge is known to impede protein aggregation.…”

“…[45,51,52] Likewise, Thal was found as a dimer in the crystal structure whereas it exists as a monomer in solution. [28] ESI-MS measurement performed under native conditions showed that Thal is present in solution both as a homodimer and as a monomer. These findings corroborate that Thal tends to dimer formation in solution similar to other halogenases.…”

“…enabled a pronounced alteration of regioselectivity that was exemplified for the Trp 6‐halogenase Thal. Exchange of merely five active‐site residues of Thal into the corresponding counterparts of RebH generated a quintuple variant exhibiting a nearly complete switch from C6‐ to C7‐halogenation with >95% selectivity …”

Structure and Activity of the Thermophilic Tryptophan‐6 Halogenase BorH

“…[20] The most extensively studied halogenases (EC 1.14.19.9, 1.14.19.58, 1.14.19.59) regioselectivity halogenate l-tryptophan on the 5, 6, or 7p ositions of the indole ring, though most have the ability to halogenate other substrates as well. [21][22][23][24][25][26][27][28] An ewly identified viral FDH has recently been shown to have iodination activity. [29] Borregomycin A( Scheme 1A), ac hlorinated bisindole alkaloid produced by oxidative dimerization of Trp, was discovered through metagenomic screening of soil samples from the Anza-Borrego desert and has antiproliferative activity against humanc olon cancer HCT116 cells and inhibits the kinase CaM-KIId.…”