Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

Nonclassical protein secretion by Bacillus subtilis in the stationary phase is not due to cell lysis.

The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic α-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.

Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11.

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.

Osmotic shock: a mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins.

When over-expressed in the cytoplasm of Escherichia coli, carboxylesterase Est55 of Geobacillus stearothermophilus was found to be released from cells upon osmotic shock. Comparing two osmotic shock protocols showed that release of Est55 was abolished in the absence of mechanosensitive channel MscL by one method but not the other. The discrepancy extended to several previously reported cytoplasmic proteins released by osmotic shock, including: EF-Tu, thioredoxin, and DnaK in E. coli. Stepwise analyses of parameters between these two protocols revealed that the use of mechanical pipetting instead of gentle dilution of cells prior to exposure to hypotonic solution abolished the effect of MscL. Furthermore, while this phenomenon of release of certain cytoplasmic proteins was sustained in all three wild type strains of E. coli, presence of gadolinium was able to serve as an MscL channel blocker and prevented release of Est55 and EF-Tu in the process. An optimized protocol of osmotic shock was developed from this study to provide a more reliable assessment of location of proteins in E. coli. This method allowed release of authentic periplasmic MalE and beta-lactamase proteins comparable to that by EDTA-lysozyme treatment.

Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30.

Est30 is a thermophilic carboxylesterase cloned from Geobacillus stearothermophilus that showed optimal hydrolysis of esters with short acyl chains at 70 degrees C. Est30 is a member of a new family of carboxylesterases with representatives in other Gram-positive bacteria. The crystal structure has been determined at 1.63A resolution using multiple anomalous dispersion data. The two-domain crystal structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. The catalytic triad consists of residues Ser94, Asp193, and His223. A 100Da tetrahedral ligand was observed to be covalently bound to the side-chain of Ser94. The propyl acetate ligand represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily.

Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus.

Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries.

Crystallization and preliminary X-ray diffraction data for the carboxylesterase Est30 from Bacillus stearothermophilus.

Crystals have been grown of the carboxylesterase Est30 from Bacillus stearothermophilus by hanging-drop vapor diffusion using ammonium sulfate as precipitant. The crystals diffracted to better than 2.0 A resolution. X-ray diffraction data were reduced in space group C222(1), with unit-cell parameters a = 55.83, b = 58.15, c = 179.65 A. R(merge) was 0.038 for 17 449 independent reflections with a completeness of 85.1%. V(M) was calculated to be 2.43 A(3) Da(-1), which suggested that there was one molecule of Est30 in the asymmetric unit. These crystals are suitable for structure determination.

Molecular cloning and characterization of the Salmonella enterica Serovar Paratyphi B rma Gene, which confers multiple drug resistance in Escherichia coli.

A genomic library from a strain of Salmonella enterica serovar Paratyphi B that exhibits multiple drug resistance (MDR) was constructed in Escherichia coli. Two of the recombinant plasmids, pNOR5 and pNOR5, conferred resistance only to fluoroquinolones in E. coli, whereas the third, pNCTR4, conferred the MDR phenotype. Sequence and subcloning analysis showed that it is the presence of RecA on the first two plasmids which confers resistance to fluoroquinolones in E. coli. A similar analysis established that the MDR phenotype conferred by pNCTR4 is due to a gene, rma (resistance to multiple antibiotics), which encodes a 13.5-kDa polypeptide. The derived sequence for Rma exhibits a high degree of similarity to those of a group of MarA-like activators that confer MDR in E. coli. A MalE-Rma fusion protein was purified to near homogeneity and was shown to interact with a DNA fragment carrying a MarA operator sequence. Furthermore, overexpression of rma in E. coli caused changes in the outer membrane protein profile that were similar to those reported for MarA. These results suggest that Rma might act as a transcriptional activator of the marA regulon.