Arthur Oubrie

Nitric oxide reductases in bacteria.

Nitric oxide reductases (NORs) that are found in bacteria belong to the large enzyme family which includes cytochrome oxidases. Two types of bacterial NORs have been characterised. One is a cytochrome bc-type complex (cNOR) that receives electrons from soluble redox protein donors, whereas the other type (qNOR) lacks the cytochrome c component and uses quinol as the electron donor. The latter enzyme is present in several pathogens that are not denitrifiers. We summarise the current knowledge on bacterial NORs, and discuss the evolutionary relationship between them and cytochrome oxidases in this review.

Structure and mechanism of soluble quinoprotein glucose dehydrogenase

Soluble glucose dehydrogenase (s‐GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism of PQQ‐dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X‐ray structure of s‐GDH with the cofactor at 2.2 Å resolution, and of a complex with reduced PQQ and glucose at 1.9 Å resolution. These structures reveal the active site of s‐GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ‐dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base‐catalyzed hydride transfer, rather than the generally accepted covalent addition‐elimination mechanism. Thus, PQQ‐dependent enzymes use a mechanism similar to that of nicotinamide‐ and flavin‐dependent oxidoreductases.

Structure and mechanism of soluble glucose dehydrogenase and other PQQ-dependent enzymes.

This paper discusses recent X-ray structures of several pyrroloquinoline quinone (PQQ)-dependent proteins in relation to their proposed modes of action. In addition, a detailed analysis of redox-related structural changes in the soluble PQQ-dependent glucose dehydrogenase is presented. A sequence comparison of that enzyme with a number of homologues shows that PQQ-dependent enzymes are much more widespread than has been assumed so far. In particular, the presence of a PQQ-dependent enzyme in at least one archaeon opens up the possibility that PQQ has been involved in prokaryotic metabolism since the early days of the evolution of bacterial life on earth.

Molecular basis for specificity of the extracytoplasmic thioredoxin ResA

ResA, an extracytoplasmic thioredoxin from Bacillus subtilis, acts in cytochrome c maturation by reducing the disulfide bond present in apocytochromes prior to covalent attachment of heme. This reaction is (and has to be) specific, as broad substrate specificity would result in unproductive shortcircuiting with the general oxidizing thioredoxin(s) present in the same compartment. Using mutational analysis and subsequent biochemical and structural characterization of active site variants, we show that reduced ResA displays unusually low reactivity at neutral pH, consistent with the observed high pKa values >8 for both active site cysteines. Residue Glu80 is shown to play a key role in controlling the acid-base properties of the active site. A model in which substrate binding dramatically enhances the reactivity of the active site cysteines is proposed to account for the specificity of the protein. Such a substratemediated activation mechanism is likely to have wide relevance for extracytoplasmic thioredoxins.

Soluble Aldose Sugar Dehydrogenase from Escherichia coli A HIGHLY EXPOSED ACTIVE SITE CONFERRING BROAD SUBSTRATE SPECIFICITY

A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The β-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.

Structural basis for agonism and antagonism for a set of chemically related progesterone receptor modulators

The progesterone receptor is able to bind to a large number and variety of ligands that elicit a broad range of transcriptional responses ranging from full agonism to full antagonism and numerous mixed profiles inbetween. We describe here two new progesterone receptor ligand binding domain x-ray structures bound to compounds from a structurally related but functionally divergent series, which show different binding modes corresponding to their agonistic or antagonistic nature. In addition, we present a third progesterone receptor ligand binding domain dimer bound to an agonist in monomer A and an antagonist in monomer B, which display binding modes in agreement with the earlier observation that agonists and antagonists from this series adopt different binding modes.

X-ray Structures of Progesterone Receptor Ligand Binding Domain in Its Agonist State Reveal Differing Mechanisms for Mixed Profiles of 11β-Substituted Steroids

Background: Understanding the molecular basis for the mixed profiles of progesterone receptor (PR) ligands will benefit future drug design. Results: Two differing mechanisms for the induction of mixed profiles by 11β-steroids are described. Conclusion: Subtle electrostatic and steric factors explain the differing PR activities of 11β-steroids. Significance: These observations will impact future drug-design strategies for PR and potentially other nuclear receptors. We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu723. This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp755, resulting in a decreased steric clash with Met909 but inducing a new internal clash with Val912 in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11β substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.

The role of ResA in type II cytochrome c maturation

Numerous bacterial proteins involved in the nitrogen cycle, and other processes, require c-type haem as a cofactor. c-type cytochromes are formed by covalent attachment of haem to the conserved CXXCH motif. Here, we briefly review what is presently known about cytochrome c maturation in Bacillus subtilis with particular emphasis on the crystal structures of ResA.

High-resolution crystal structures of factor XIa coagulation factor in complex with nonbasic high-affinity synthetic inhibitors.

Factor XI (FXI) is a key enzyme in the coagulation pathway and an attractive target for the development of anticoagulant drugs. A small number of high-resolution crystal structures of FXIa in complex with small synthetic inhibitors have been published to date. All of these ligands have a basic P1 group and bind exclusively in the nonprime side of the active site of FXIa. Here, two structures of FXIa in complex with nonbasic inhibitors that occupy both the prime and nonprime sides of the active site are presented. These new structures could be valuable in the design and optimization of new FXIa synthethic inhibitors.

Structure of the Membrane-intrinsic Nitric Oxide Reductase from Roseobacter denitrificans.

Membrane-intrinsic nitric oxide reductases (NORs) are key components of bacterial denitrification pathways with a close evolutionary relationship to the cytochrome oxidase (COX) complex found in aerobic respiratory chains. A key distinction between COX and NOR is the identity of the metal directly opposite heme b3 within the active site. In NOR, this metal is iron (FeB), whereas in COX, it is copper (CuB). The purified NOR of Roseobacter denitrificans contains copper and has modest oxidase activity, raising the possibility that a COX-like active site might have independently arisen within the context of a NOR-like protein scaffold. Here we present the crystal structure of the Roseobacter denitrificans NorBC complex and anomalous scattering experiments probing the identity of each metal center. Our results refute the hypothesis that copper occupies the active site and instead reveal a new metal center in the small subunit not seen in any other NOR or COX.