Not cleaving the his-tag of thal results in more tightly packed and better-diffracting crystals

Abstract

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2 ), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21 ) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.