Crystal structures reveal metal-binding plasticity at the metallo-β-lactamase active site of PqqB from Pseudomonas putida

Abstract

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone (PQQ) and a distal member of the metallo-β-lactamase (MBL) superfamily. PqqB lacks two residues in the conserved signature motif HxHxDH that makes up the key metal-chelating elements that can bind up to two metal ions at the active site of MBLs and other members of its superfamily. Here we report crystal structures of PqqB bound to Mn2+, Mg2+, Cu2+ and Zn2+. These structures demonstrate that PqqB can still bind metal ions at the canonical MBL active site. The fact that PqqB can adapt its side chains to chelate a wide spectrum of metal ions with different coordination features on a uniform main chain scaffold demonstrates its metal binding plasticity. This plasticity may provide insights into the structural basis of promiscuous activities found in ensembles of metal complexes within this superfamily. Furthermore, PqqB belongs to a small subclass of MBLs that contain an additional CxCxxC motif that binds a structural Zn2+. Our data support a key role for this motif in dimerization.

New developments in RiPP discovery, enzymology and engineering

“…60C). 258,259 Subsequently, a cyclization step that is postulated to be spontaneous 258 provides the last intermediate that is oxidized by PqqC in an eight electron oxidation process that does not require any cofactors. 260 Thus, in recent years the enzymatic activities of PQQ biosynthesis have been elucidated, either with authentic substrates or substrate analogs.…”

Discovery of Hydroxylase Activity for PqqB Provides a Missing Link in the Pyrroloquinoline Quinone Biosynthetic Pathway

“…However, all efforts to establish a robust, metal-dependent reaction of PqqB with Cys-Tyr were unsuccessful, with the notable exception of an Fe 2+ -catalyzed oxidative decarboxylation of alpha-ketoglutarate (α-KG) (Figure a and b and Figure S6). The suggestion that α-KG, a common cosubstrate of nonheme iron hydroxylases, could act as a redox cosubstrate in PqqB came from the X-ray structure of the Mn 2+ -reconstituted enzyme from P. putida , which required malic acid to retain significant bound metal in the crystal . Addition of Cys-Tyr and α-KG to PqqB does in fact give rise to a slow increase in absorbance between 350 and 550 nm (Figure a), although the very low level of product precluded detailed characterization.…”

“…PqqB has been reported to hydrolyze amide and thioester bonds of the substrates of MBL and glyoxalase, respectively; however, the turnover rates for these processes are extremely low and suggest residual ancestral activity. Without a conclusive demonstration of robust PqqB activity, proposals of function remain speculative. ,, Herein, we present a combination of metal and ligand binding studies, X-ray crystallography, and detailed activity assays to show that PqqB is a previously uncharacterized hydroxylase. The aggregate data indicate that PqqB is capable of generating a hydroxyquinone intermediate that possesses the final quinone moiety of the mature PQQ cofactor.…”

“…PqqB belongs to the metallo β-lactamase (MBL) family and contains two metal binding sites per monomer; one of these contains a tightly bound structural Zn 2+ that induces correct protein folding and dimerization . The second metal binding site, and putative active site, presents a 2-His/1-Asp ligand set for metal coordination (see Supporting Information, Figure S1).…”

Occurrence, function, and biosynthesis of mycofactocin

“…Gliese et al 2010). Next, it has been proposed that PqqB oxidizes the crosslinked diamino acid to form the trihydroxy-phenylalanine (Shen et al 2012;Tu et al 2017). The spontaneous oxidation of the trihydroxyphenyalanine could lead to a nucleophilic substitution and cyclization reaction involving the free amino group of glutamate to the para position of trihydroxyphenylalanine leading to the intermediate 3a-(2-amino-2carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ).…”