Structural Insights from Molecular Dynamics Simulations of Tryptophan 7-Halogenase and Tryptophan 5-Halogenase

Abstract

Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and chemotherapeutics (salinosporamide A and rebeccamycin) are chlorinated. Halogenating enzymes like tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH) perform regioselective halogenation of tryptophan. In this study, the conformational dynamics of two flavin-dependent tryptophan halogenases—PrnA and PyrH—was investigated through molecular dynamics simulations, which are in agreement with the crystallographic and kinetic experimental studies of both enzymes and provide further explanation of the experimental data at an atomistic level of accuracy. They show that the binding sites of the cofactor-flavin adenine dinucleotide and the substrate do not come into close proximity during the simulations, thus supporting an enzymatic mechanism without a direct contact between them. Two catalytically important active site residues, glutamate (E346/E354) and lysine (K79/K75) in PrnA and PyrH, respectively, were found to play a key role in positioning the proposed chlorinating agent, hypochlorous acid. The changes in the regioselectivity between PrnA and PyrH arise as a consequence of differences in the orientation of substrate in its binding site.

Genomic Determinants Encode the Reactivity and Regioselectivity of Flavin-Dependent Halogenases in Bacterial Genomes and Metagenomes

“…The discrete chemical nature of BrO − and ClO − and/or kinetic and/or thermodynamic parameters in transfer reactions of hypohalite to tryptophan or the indole-binding region via catalytic lysine might be in part responsible for the determination of the reactivities. The latter is consistent with the molecular dynamics (MD) simulation studies of PrnA and PyrH ( 64 ), where significant conformational changes occur in the transfer of hypohalite. MHal1 was inactive even with indole, which is presumably related to the substantially altered sequence nearby the Trp-binding region (see Fig.…”

Conformational flexibility influences structure–function relationships in nucleic acidN-methyl demethylases

“…Related computational strategies have been successfully implemented for investigating structure-function relationships of various other enzymes. [28][29][30][31][32][33] The results show conformational dynamics exercise distinct effects in AlkB and FTO and are important in determining substrate selectivity. Specific residues in AlkB/FTO play important roles in correlated motions/flexibility and are important in maintaining the productive geometry of reactant complexes.…”

Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals

“…[20] The specific roles played by structural dynamics and active site residues in the regio selective halogenation mechanism were being investigated by several recent computational and spectroscopic studies. [19,110,111] The quantum mechanics (QM)/molecular mechanics (MM) approaches supported the key atomistic interactions in the reaction path confirming the respective proton acceptor and donor roles of active site lysine and glutamate residues. A structurally conserved and highly flexible 'strap' region, present in flavindependent tryptophan halogenases, links the FAD and substrate-binding domain and is also known to be involved in FAD interactions and catalysis.…”