Bárbara M. Calisto

Essential role of proximal histidine-asparagine interaction in mammalian peroxidases

In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.

Biosynthesis of isoprenoids in plants: Structure of the 2C-methyl-D-erithrytol 2,4-cyclodiphosphate synthase from Arabidopsis thaliana. Comparison with the bacterial enzymes

The X‐ray crystal structure of the 2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 Å resolution in complex with a cytidine‐5‐monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate‐like binding pocket, with a proposed feedback‐regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.

Re‐engineering specificity in 1,3‐1, 4‐β‐glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3‐1,4‐β‐glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16‐34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco‐oligosaccharides, the second binds branched xylogluco‐oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco‐ and linear gluco‐oligosaccharides, enzyme engineering on the negative subsites of 1,3‐1,4‐β‐glucanase to accept branched substrates has been undertaken. Removal of the 1,3‐1,4‐β‐glucanase major loop and replacement with that of XET16‐34 to open the binding cleft resulted in a folded protein, which still maintained some β‐glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3‐1,4‐β‐glucanase β‐sheets forming the binding site cleft were mutated to resemble XET16‐34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β‐glucan. Therefore, binding specificity has been re‐engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X‐ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity. Proteins 2011.

The EAGR box structure: a motif involved in mycoplasma motility

Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self‐replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell‐host adhesion. A well‐conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra‐subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra‐ and inter‐subunit symmetry axes, support a role for the EAGR box in protein–protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility.

Crystal Structure of Brucella abortus Deoxyxylulose-5-phosphate Reductoisomerase-like (DRL) Enzyme Involved in Isoprenoid Biosynthesis

Background: The current antibiotic resistance epidemic demands new drugs specifically targeting infective agents. Results: The crystal structure of the Brucella DRL enzyme shows major differences compared with DXR, which catalyzes the same reaction in most other bacteria. Conclusion: Structural information will allow development of inhibitors targeting only DRL. Significance: Drugs against DRL could function as highly specific, narrow-range antibiotics. Most bacteria use the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-d-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity.

The race to resolve the atomic structures of the ribosome: On the Nobel Prize in Chemistry awarded to Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath

The Nobel Prize in Chemistry 2009 was awarded to three scientists, Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath, for their investigations into the structure and functioning of ribosomes. These complex cellular particles are where genetic information is decoded and proteins are synthesized. Consequently, ribosomes play a central role inthe biology of all living organisms. Ribosomes are composed of one small and one large subunit, which in prokaryotes are respectively referred to as 30S and 50S according to their sedimentation properties. In both subunits, about two thirds of the mass corresponds to ribosomal RNA (rRNA) and the rest to different proteins. Given their biological relevance and the fact that they are the target of a large variety of clinically relevant antibiotics, ribosomes have been the subject of intense and continuousresearch since the 1960s, when the genetic code was unraveled. These investigations led, and to some extent culminated, with the results published in 2000 (annus mirabilis for ribosomes), reporting the crystal structures of the 50S ribosomal subunit from Haloarcula marismortui at 2.4A resolution and, a few weeks later, of the 30S subunit from Thermus thermophilus at 3.3A and 3.0A resolution, by teams led by the three laureates. These results have been instrumental in understanding ribosome function at the atomic level. However, there are many years of work ahead, as much remains to be learned about ribosomes; in particular the structure of the eukaryotic ribosome has yet to be elucidated.