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author:("sulek, Jorge")
1.  First co-expression of a lipase and its specific foldase obtained by metagenomics 
Microbial Cell Factories  2014;13(1):171.
Background
Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil.
Results
Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni2+ column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg−1, being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg−1, respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser103, Asp250, His272), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/β hydrolase folding type I.
Conclusions
This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.
doi:10.1186/s12934-014-0171-7
PMCID: PMC4305245  PMID: 25510188
Lipases; Metagenomics; Biocatalysis; Lipase-foldase co-expression
2.  Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the Leishmaniases† 
Journal of Medicinal Chemistry  2009;53(1):221-229.
Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.
doi:10.1021/jm901059x
PMCID: PMC2804273  PMID: 19916554
3.  Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive “Lid” 
PLoS ONE  2009;4(11):e8006.
Background
The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission.
Methodology
Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 Å resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date.
Conclusion
The structure of AaegOBP1 ( = AaegOBP39) shares the common fold of insect OBPs with six α-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this “lid” may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.
doi:10.1371/journal.pone.0008006
PMCID: PMC2778553  PMID: 19956631
4.  Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase complexed with 6-phosphogluconate 
The structure of 6-phosphogluconate dehydrogenase from a moderate thermophile, G. stearothermophilus, is presented and compared with those of orthologous enzymes.
Two crystal structures of recombinant Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase (Gs6PDH) in complex with the substrate 6-­phosphogluconate have been determined at medium resolution. Gs6PDH shares significant sequence identity and structural similarity with the enzymes from Lactococcus lactis, sheep liver and the protozoan parasite Trypanosoma brucei, for which a range of structures have previously been reported. Comparisons indicate that amino-acid sequence conservation is more pronounced in the two domains that contribute to the architecture of the active site, namely the N-terminal and C-terminal domains, compared with the central domain, which is primarily involved in the subunit–subunit associations required to form a stable dimer. The active-site residues are highly conserved, as are the interactions with the 6-phosphogluconate. There is interest in 6PDH as a potential drug target for the protozoan parasite T. brucei, the pathogen responsible for African sleeping sickness. The recombinant T. brucei enzyme has proven to be recalcitrant to enzyme–ligand studies and a surrogate protein might offer new opportunities to investigate and characterize 6PDH inhibitors. The high degree of structural similarity, efficient level of expression and straightforward crystallization conditions mean that Gs6PDH may prove to be an appropriate model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species.
doi:10.1107/S1744309109012767
PMCID: PMC2675582  PMID: 19407374
pentose phosphate pathway; 6-phosphogluconate dehydrogenase; Geobacillus stearothermophilus

Results 1-4 (4)