Brenda Valderrama

Suicide Inactivation of Peroxidases and the Challenge of Engineering More Robust Enzymes

As the number of industrial applications for proteins continues to expand, the exploitation of protein engineering becomes critical. It is predicted that protein engineering can generate enzymes with new catalytic properties and create desirable, high-value, products at lower production costs. Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications. However, peroxidases are unstable and are readily inactivated by their substrate, hydrogen peroxide. Researchers rely on the powerful tools of molecular biology to improve the stability of these enzymes, either by protecting residues sensitive to oxidation or by devising more efficient intramolecular pathways for free-radical allocation. Here, we discuss the catalytic cycle of peroxidases and the mechanism of the suicide inactivation process to establish a broad knowledge base for future rational protein engineering.

Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter

Pseudomonas aeruginosa contains two transcription regulators (LasR and RhlR) that, when complexed with their specific autoinducers (3-oxo-dodecanoyl-homoserine lactone and butanoyl-homoserine lactone, respectively) activate transcription of different virulence-associated traits. We studied the RhlR-dependent transcriptional regulation of the rhlAB operon encoding rhamnosyltransferase 1, an enzyme involved in the synthesis of the surfactant monorhamnolipid, and showed that RhlR binds to a specific sequence in the rhlAB regulatory region, both in the presence and in the absence of its autoinducer. Our data suggest that in the former case it activates transcription, whereas in the latter it acts as a transcriptional repressor of this promoter. RhlR seems to repress the transcription of other quorum-sensing-regulated genes; thus, RhlR repressor activity might be of importance in the finely regulated expression of P. aeruginosa virulence-associated traits.

Microsomal transformation of organophosphorus pesticides by white rot fungi

The enzymatic mechanism for the transformationof organophosphorus pesticides (OPPs) by differentwhite-rot fungi strains was studied. With theexception of Ganoderma applanatum 8168,all strains from a collection of 17 different fungicultures were able to deplete parathion. Threestrains showing the highest activities were selectedfor further studies: Bjerkandera adusta 8258,Pleurotus ostreatus 7989 and Phanerochaetechrysosporium 3641. These strains depleted 50 to96% of terbufos, azinphos-methyl, phosmet andtribufos after four-days exposure to the pesticides.In order to identify the cellular localization of thetransformation activity, the extracellular andmicrosomal fractions of Pleurotus ostreatus7989 were evaluated in vitro. While the activitiesof ligninolytic enzymes (lignin peroxidase,manganese peroxidase and laccase) weredetected in the extracellular fraction, noenzymatic modification of any of the fivepesticides tested could be found, suggestingthe intracellular origin of the transformationactivity. In accordance with this observation themicrosomal fraction was found able to transformthree OPPs with the following rates:10 μmol mg prot-1 h-1 forphosmet, 5.7 μmol mg prot-1 h-1 forterbufos, and 2.2 μmol mg prot-1 h-1 forazinphos-methyl. The products from these reactions andfrom the transformation of trichlorfon and malathion,were identified by mass-spectrometry. These results,supported by specific inhibition experiments and thestringent requirement for NADPH during the in vitroassays suggest the involvement of a cytochrome P450.

Evolutionary and structural diversity of fungal laccases

Fungal laccases have been extensively exploited for industrial purposes and there is a wealth of information available regarding their reaction mechanism, biological role and several molecular aspects, including cloning, heterologous expression and transcriptional analyses. Here we present the reconstruction of the fungal laccase loci evolution inferred from the comparative analysis of 48 different sequences. The topology of the phylogenetic trees indicate that a single monophyletic branch exists for fungal laccases and that laccase isozyme genes may have evolved independently, possibly through duplication-divergence events. Laccases are copper-containing enzymes generally identified by the utilization of substituted p-diphenol substrates. Interestingly, our approach permitted the assignment of two copper-containing oxidases, preliminarily catalogued as laccases, to a different evolutionary group, distantly related to the main branch of bona fide laccases.

Rhizobium etli genetically engineered for the heterologous expression of Vitreoscilla sp. hemoglobin : Effects on free-living and symbiosis

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibi...

Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase

X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV-Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O(2) reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account.

Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering

Iso‐1‐cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox‐inspired strategy was implemented until an iso‐1‐cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15‐fold higher total‐turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein‐based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox‐inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.—Valderrama, B., García‐Arellano, H., Giansanti, S., Baratto, M. C., Pogni, R., Vazquez‐Duhalt, R. Oxidative stabilization of iso‐1‐cytochrome c by redox‐inspired protein engineering. FASEB J. 20, E472–E481 (2006)

Two mammalian glucosamine-6-phosphate deaminases: a structural and genetic study

Glucosamine‐6‐phosphate deaminase (EC 3.5.99.6) is an allosteric enzyme that catalyzes the reversible conversion of D‐glucosamine‐6‐phosphate into D‐fructose‐6‐phosphate and ammonium. Here we describe the existence of two mammalian glucosamine‐6‐phosphate deaminase enzymes. We present the crystallographic structure of one of them, the long human glucosamine‐6‐phosphate deaminase, at 1.75 Å resolution. Crystals belong to the space group P212121 and present a whole hexamer in the asymmetric unit. The active‐site lid (residues 162–182) presented significant structural differences among monomers. Interestingly the region with the largest differences, when compared with the Escherichia coli homologue, was found to be close to the active site. These structural differences can be related to the kinetic and allosteric properties of both mammalian enzymes.

Transcriptional activity of the symbiotic plasmid of Rhizobium etli is affected by different environmental conditions

Global patterns of transcriptional activity of the symbiotic plasmid (pSym) of Rhizobium etli were studied under a variety of environmental conditions, including some relevant to the symbiotic process. 32P-labelled single-stranded complementary DNAs synthesized from total RNA were used as hybridization probes against an ordered collection of cosmid clones that covered the whole pSym. Our results showed that, under aerobic conditions, discrete regions of the pSym are differentially transcribed depending on the carbon and nitrogen sources employed. In general, poor carbon or nitrogen sources allowed greater expression than rich ones. Time-course experiments with the nod gene inducer genistein led us to the identification of new regions responsive to this flavonoid. Widespread transcription was observed during microaerobiosis, but not under aerobic conditions, indicating that oxygen concentration is a major effector of transcriptional activity in the pSym. This response was reduced, but not suppressed, in a nifA mutant, indicating the location of regions whose transcription may depend on other oxygen-sensitive regulators. During symbiosis, almost the entire pSym was actively transcribed and the transcription pattern was similar to that observed during microaerobiosis. The experimental approach described allowed the identification and localization of specific regions in the pSym whose expression depends on defined environmental stimuli.

Evolution and diversity of periplasmic proteins involved in copper homeostasis in gamma proteobacteria

BackgroundDifferent systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components.ResultsBy the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires.ConclusionsThese observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems.