Molecular basis of enzymatic nitrogen-nitrogen formation by a family of zinc-binding cupin enzymes

Abstract

Molecules with a nitrogen-nitrogen (N-N) bond in their structures exhibit various biological activities and other unique properties. A few microbial proteins are recently emerging as dedicated N-N bond forming enzymes in natural product biosynthesis. However, the details of these biochemical processes remain largely unknown. Here, through in vitro biochemical characterization and computational studies, we report the molecular basis of hydrazine bond formation by a family of di-domain enzymes. These enzymes are widespread in bacteria and sometimes naturally exist as two standalone enzymes. We reveal that the methionyl-tRNA synthase-like domain/protein catalyzes ATP-dependent condensation of two amino acids substrates to form a highly unstable ester intermediate, which is subsequently captured by the zinc-binding cupin domain/protein and undergoes redox-neutral intramolecular rearrangement to give the N-N bond containing product. These results provide important mechanistic insights into enzymatic N-N bond formation and should facilitate future development of novel N-N forming biocatalyst.

Synthetic and biosynthetic routes to nitrogen–nitrogen bonds

“…To date, several enzymes have been definitively linked to N–N bonds-forming reactions through in vitro studies, including in the biosynthesis of piperazic acid 58 and in the construction of nitrosamines. 147 For the biosynthesis of linear hydrazines, a family of zinc-binding cupin protein/domain has been shown to be responsible for the N–N bond formation in several different molecules; 80,88,116 in alazopeptin biosynthesis, AzpL has been linked to diazo formation; 118 and emerging data shows the critical role of the ATP-dependent Tri17 in N -nitrosation to give triascins. 116 A combination of enzyme-catalyzed and spontaneous reactions appear to form N–N bonds in cremeomycins, 110,115 xiamycins, 95,97 azoxymycins, 191 and 8-azaguanine.…”

“…Both cupin and MetRS domains appeared to be required for the production of hydrazine product 112 , as shown in a Tri26-28 coupled assays to generate 114 , which is consistent with the results from other PyrN homologs. 88 Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis also revealed each domain of Tri28 coordinates one zinc. In the biosynthesis of triacsins, 114 was succinylated by Tri31 to form 333 and then ATP-dependent synthetase Tri29 transferred it onto one ACP Tri30.…”

“…81 Recently, Du et al elucidated the catalytic mechanism of PyrN. 88 They first incubated PyrN with L-Glu, synthesized N 6 -OH-L-lysine 110, ATP, Mg 2+ , glutamyl-tRNA or total aa-tRNA mixture from E. coli, and the hydrazine product 117 was successfully detected in the reactions. When glutamyl-tRNA/ total aa-tRNA mixture was removed from the assays, 117 was still formed, but removal of ATP, Mg 2+ , or PyrN abolished N-N bond formation.…”

“…The results also revealed the MetRS domains/stand-alone proteins displayed strict substrate specificities, but some cupin domains/proteins showed flexible substrate selectivity and can accept different N-O intermediates generated in situ from coupled MetRS assays to afford hydrazines. 88…”

“…73 The leading method used for pyrazole derivative synthesis is through condensation of hydrazines 92 and 1,3-difunctional compounds. 88–91 1,3-Dipolar cycloaddition of diazoalkanes 95 with alkenes or alkynes 94 and some multicomponent reactions can also be used to acquire pyrazole derivatives. Those approaches tend to employ intact N–N substrates, like hydrazine or diazos, to build pyrazole heterocycles (Fig.…”