The structural basis of pyridoxal-5′-phosphate-dependent β-NAD-alkylating enzymes
Takayoshi Awakawa, Takahiro Mori, Lena Barra, Yusef Ahmed, Richiro Ushimaru, Yaojie Gao, Naruhiko Adachi, Toshiya Senda, Tohru Terada, Dean J. Tantillo, Ikuro Abe
Abstract
SbzP is a unique pyridoxal-5′-phosphate-dependent enzyme, which catalyses a [3+2] annulation between the pyridinium ring of β-nicotinamide adenine dinucleotide (β-NAD) and an electron rich β,γ-unsaturated quinonoid derived from S-adenosylmethionine in natural product azaindane antibiotics biosynthesis. The SbzP-mediated annulation has been proposed to be a rare tandem C–C bond formation, but its structural basis and catalytic mechanism remain largely unknown. Here we report the β-NAD-complexed structure of PseP (SbzP homologue), identified by cryo-electron microscopy. Structure-based mutagenesis, stopped-flow analysis, thermal shift and surface plasmon resonance analysis identified the important residues for the substrate binding. Molecular dynamics simulations provided insights regarding how the enzyme orients the Cγ of the unsaturated quinonoid to β-NAD. In addition, density functional theory calculations confirmed that the proposed stepwise mechanism is more likely than a pericyclization mechanism. This study provides the structural basis of a pyridoxal-5′-phosphate-dependent enzyme that catalyses nucleophilic Cγ addition and β-NAD processing in natural product biosynthesis. Recently, the pyridoxal-5′-phosphate-dependent enzyme SbzP was reported to catalyse a [3+2]-annulation reaction yielding β-NAD-derived antibiotics. Now, cryo-electron microscopy structures of a stable homologue and computational simulations provide structural and mechanistic insights into this enzymatic reaction.