Karl A. P. Payne

Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon–cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen–cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor–cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1′ bond breakage following FMN reduction, leading to formation of the N5–C1′ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3′–C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding.

Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM–MS data.

Oxidative Maturation and Structural Characterization of Prenylated FMN Binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis.

The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained. Formation of holoUbiD requires reconstitution in vitro of the apoUbiD with reduced prFMN. Furthermore, although the Fdc1 apoenzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holoUbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holoUbiD reveal a relatively open active site potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals an unexpected variation occurs in domain mobility, prFMN binding, and maturation by the UbiD enzyme family.

Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification

Background: Little is known about epoxyqueuosine reductase (QueG), which catalyzes the final step in the biosynthesis of queuosine. Results: We report solution and structural characterization of Streptococcus thermophilus QueG. Conclusion: The QueG similarity to reductive dehalogenases is largely limited to cofactor binding. Significance: Our study establishes the link between cobalamin-metabolism and tRNA modification and suggests a mechanism for cobalamin-dependent epoxide reduction. Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5′-GUN-3′ tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes.

Preservation of human tear protein structure and function by a novel contact lens multipurpose solution containing protein-stabilizing agents.

Objectives: Tear film proteins have antimicrobial and other functions that may be lost after denaturation during contact lens wear. A new multipurpose solution has recently become available (Biotrue, Bausch + Lomb Inc., Rochester, NY), which contains protein-stabilizing agents including hyaluronic acid, poloxamine, and sulfobetaine 10, the latter used previously as a laboratory tool to renature proteins. We examine whether this new multipurpose solution formulation can prevent the denaturation of human lactoferrin and lysozyme at physiologic levels in response to a powerful denaturing challenge. Methods: Human lactoferrin and lysozyme were treated with sodium dodecyl sulfate (SDS) either with or without an investigational version of the new multipurpose solution (without its two disinfectant agents) (investigational multipurpose solution [iMPS]). The structure was assessed by native-polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorometry; additionally, antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus was measured. Results: The iMPS prevented an SDS-induced shift in the native-PAGE banding position of lactoferrin. The SDS treatment substantially altered the lactoferrin DSC and fluorescence spectra, indicating that the protein had denatured. This change did not occur in the presence of iMPS. Lactoferrin and lysozyme showed antibacterial and bacteriolytic activity, which was abolished after SDS treatment; this loss of activity did not occur for proteins treated with iMPS. Conclusions: These data clearly show that the iMPS prevents the denaturation of physiologic levels of human lactoferrin and lysozyme by the strongly denaturing surfactant SDS and that stabilized proteins retain their function. We conclude that this solution has the capacity to stabilize the structure and function of tear proteins.

The UbiX-UbiD system: The biosynthesis and use of prenylated flavin (prFMN)

The UbiX-UbiD system consists of the flavin prenyltransferase UbiX that produces prenylated FMN that serves as the cofactor for the (de)carboxylase UbiD. Recent developments have provided structural insights into the mechanism of both enzymes, detailing unusual chemistry in each case. The proposed reversible 1,3-dipolar cycloaddition between the cofactor and substrate serves as a model to explain many of the key UbiD family features. However, considerable variation exists in the many branches of the UbiD family tree.

Discovery of a putative acetoin dehydrogenase complex in the hyperthermophilic archaeon Sulfolobus solfataricus.

Like many other aerobic archaea, the hyperthermophile Sulfolobus solfataricus possesses a gene cluster encoding components of a putative 2‐oxoacid dehydrogenase complex. In the current paper, we have cloned and expressed the first two genes of this cluster and demonstrate that the protein products form an α2β2 hetero‐tetramer possessing the catalytic activity characteristic of the first component enzyme of an acetoin dehydrogenase multienzyme complex. This represents the first report of an acetoin multienzyme complex in archaea, and contrasts with the branched‐chain 2‐oxoacid dehydrogenase complex activities characterised in two other archaea, Thermoplasma acidophilum and Haloferax volcanii.

Crystal structure of X-prolyl aminopeptidase from Caenorhabditis elegans: A cytosolic enzyme with a di-nuclear active site.

Eukaryotic aminopeptidase P1 (APP1), also known as X‐prolyl aminopeptidase (XPNPEP1) in human tissues, is a cytosolic exopeptidase that preferentially removes amino acids from the N‐terminus of peptides possessing a penultimate N‐terminal proline residue. The enzyme has an important role in the catabolism of proline containing peptides since peptide bonds adjacent to the imino acid proline are resistant to cleavage by most peptidases. We show that recombinant and catalytically activeCaenorhabditis elegans APP‐1 is a dimer that uses dinuclear zinc at the active site and, for the first time, we provide structural information for a eukaryotic APP‐1 in complex with the inhibitor, apstatin. Our analysis reveals thatC. elegans APP‐1 shares similar mode of substrate binding and a common catalytic mechanism with other known X‐prolyl aminopeptidases.