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Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo β-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.

PqqB is an enzyme involved in the biosynthesis of pyrroloquinoline quinone 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.

Biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is initiated when the precursor peptide, PqqA, is recognized and bound by the RiPP precursor peptide recognition element (RRE), PqqD, for presentation to the first enzyme in the pathway, PqqE. Unlike other RiPP-producing, postribosomal peptide synthesis (PRPS) pathways in which the RRE is a component domain of the first enzyme, PqqD is predominantly a separate scaffolding protein that forms a ternary complex with the precursor peptide and first tailoring enzyme. As PqqD is a stable, independent RRE, this makes the PQQ pathway an ideal PRPS model system for probing RRE interactions using nuclear magnetic resonance (NMR). Herein, we present both the solution NMR structure of Methylobacterium extorquens PqqD and results of 1H-15N HSQC binding experiments that identify the PqqD residues involved in binding the precursor peptide, PqqA, and the enzyme, PqqE. The reported structural model for an independent RRE, along with the mapped binding surfaces, will inform future efforts both to understand and to manipulate PRPS pathways.

The ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is a dehydrogenase cofactor synthesized by, but not exclusively used by, certain prokaryotes. RiPPs represent a rapidly expanding and diverse class of natural products-many of which have therapeutic potential-and the biosynthetic pathways for these are gaining attention. Five gene products from the pqq operon (PqqA, PqqB, PqqC, PqqD, and PqqE) are essential for PQQ biosynthesis. The substrate is the peptide PqqA, which is presented to the radical SAM enzyme PqqE by the small protein PqqD. PqqA is unstructured in solution, and only binds to PqqE when in complex with PqqD. PqqD is a member of a growing family of RiPP chaperone proteins (or domains in most cases) that present their associated peptide substrates to the initial RiPP biosynthesis enzymes. An X-ray crystal structure exists for dimeric Xanthomonas campestris PqqD (PDB ID: 3G2B), but PqqD is now known to act as a monomer under physiological conditions. In this study, the PqqD truncation from naturally fused Methylobacterium extorquens (Mex) PqqCD was overexpressed in Escherichia coli and MexPqqA was chemically synthesized. Solution NMR (1)H-,(15)N-HSQC chemical shift studies have identified the PqqD residues involved in binding PqqA, and (1)H, (13)C, and (15)N peak assignments for PqqD alone and for PqqD bound to PqqA are reported herein.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O2 activation between the aminoquinol and Cu(I).

A new experimental system that allows direct continuous monitoring of MauG-dependent tryptophan tryptophylquinone (TTQ) biosynthesis in vitro and detailed study of factors that is required for MauG-dependent TTQ biosynthesis is described. Results reveal that along with the incorporation of oxygen into the monohydroxylated biosynthetic intermediate, the subsequent oxidation of quinol, methylamine dehydrogenase (MADH) during TTQ biosynthesis also constitutes a MauG-dependent process.

A comparative structural, kinetic, and spectral analysis of the wild-type Escherichia coli copper amine oxidase and the variant Y369F indicate that tyrosine-369 mutation causes only moderate changes in the kinetic parameters of the enzyme, suggesting that the tyrosine hydroxyl is compensated within the active site.

A crystallographic structural analysis with subsequent mutagenesis study of the complex composed of the inhibitor 2-hydrazinopyridine and Escherichia coli amine oxidase (ECAO) was done to elucidate the identity of the catalytic base and the mode of substrate binding. Asp 383 was identified as the catalytic general base. Substrate binding, on the other hand, was observed to occur at the tip of the pyridine ring located at the molecular interface between D3 and D4. Fuller understanding of the activities of ECAO can be achieved with further studies about the redox chemistry involved.

The crystal structure of diheme enzyme Y294H MauG in complex with precursor protein of methylamine dehydrogenase (preMADH) was determined to the understand His-His axial ligation observed in heme MauG when Tyr294, the unusual distal axial ligand of one c-type heme was mutated to His. The mutation was found to affect not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 [Angstrom] separation of the heme iron centers.

The structural role of coenzyme B (CoBSH) early in the methyl-coenzyme M reductase (MCR) mechanism is understood by determining crystal structures of MCR is complex with four different CoBSH analogues. The analogues have reacted with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis.

The X-ray crystal structure and steady state kinetic studies were performed to examine the substrate specificity of two copper amine oxidases from Hansenula polymorpha (HPAO-1 and HPAO-2). The enzyme with the smaller substrate binding pocket in HPAO-1, an increase in substrate size affected both initial substrate binding and proton loss while in the HPAP-2 enzyme with the larger substrate binding pocket, the rate of proton loss is differentially affected when a phenyl substituent in the substrate is reduced to the size of a methyl group.

The X-ray crystal structure of a monoheme c-type cytochrome, cytochrome P460, from Nitrosomonas europea, is determined. The results have presented a novel [beta]-sheet-containing homodimeric cytochrome fold, as well as the lysine-porphyrin cross-link, whose nature has differed unexpectedly from the equivalent cross-link in hydroxylamine oxidoreductase (HAO).

The order of oxygen incorporation into the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase is investigated by incubating the monohydroxylated methylamine dehydrogenase (MADH) biosynthetic intermediate isolated in TTQ synthesis, which lacks the crosslink, in vitro with purified MauG, MADH and either isotopically labeled water for 24 hours. The data obtained indicate that after translocation into the periplasm and formation of the six-disulfide linkages, the first MauG-independent hydroxylation of betaW57 occurs specifically at the C7 position in a [beta]D76-dependent manner.

The isolated monohydroxylated biosynthetic intermediate is incubated in vitro with purified MauG to determine whether the latter is competent to catalyze the remaining steps in tryptophan tryptophylquinone (TTQ) biosynthesis. The results indicate that, in the presence of MauG, incorporation of the second oxygen into betaTrp and formation of the cross-link with betaTrp to form TTQ occurs in vitro.