A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism.     

Breast cancer in the previously augmented breast

Breast cancer in a previously augmented breast raises questions regarding cancer detection and staging, surgical and adjuvant treatment options, reconstructive outcomes, management of the contralateral breast, and continued breast cancer surveillance. This article explores the oncologic and reconstructive issues relevant to women desiring cosmetic breast implants and women with breast cancer who have undergone prior cosmetic breast augmentation.     

Sulfhydryl oxidation, not disulfide isomerization, is the principal function of protein disulfide isomerase in yeast Saccharomyces cerevisiae

Protein disulfide isomerase (PDI) is an essential protein folding assistant of the eukaryotic endoplasmic reticulum that catalyzes both the formation of disulfides during protein folding (oxidase activity) and the isomerization of disulfides that may form incorrectly (isomerase activity). Catalysis of thiol-disulfide exchange by PDI is required for cell viability in Saccharomyces cerevisiae, but there has been some uncertainty as to whether the essential role of PDI in the cell is oxidase or isomerase. We have studied the ability of PDI constructs with high oxidase activity and very low isomerase activity to complement the chromosomal deletion of PDI1 in S. cerevisiae. A single catalytic domain of yeast PDI (PDIa') has 50% of the oxidase activity but only 5% of the isomerase activity of wild-type PDI in vitro. Titrating the expression of PDI using the inducible/repressible GAL1-10 promoter shows that the amount of wild-type PDI protein needed to sustain a normal growth rate is 60% or more of the amount normally expressed from the PDI1 chromosomal location. A single catalytic domain (PDIa') is needed in molar amounts that are approximately twice as high as those required for wild-type PDI, which contains two catalytic domains. This comparison suggests that high (>60%) PDI oxidase activity is critical to yeast growth and viability, whereas less than 6% of its isomerase activity is needed.     

Zinc-dependent dimerization of the folding catalyst, protein disulfide isomerase

Protein disulfide isomerase (PDI), an essential folding catalyst and chaperone of the endoplasmic reticulum (ER), has four structural domains (a-b-b'-a'-) of approximately equal size. Each domain has sequence or structural homology with thioredoxin. Sedimentation equilibrium and velocity experiments show that PDI is an elongated monomer (axial ratio 5.7), suggesting that the four thioredoxin domains are extended. In the presence of physiological levels (<1 mM) of Zn(2+) and other thiophilic divalent cations such as Cd(2+) and Hg(2+), PDI forms a stable dimer that aggregates into much larger oligomeric forms with time. The dimer is also elongated (axial ratio 7.1). Oligomerization involves the interaction of Zn(2+) with the cysteines of PDI. PDI has active sites in the N-terminal (a) and C-terminal (a')thioredoxin domains, each with two cysteines (CGHC). Two other cysteines are found in one of the internal domains (b'). Cysteine to serine mutations show that Zn(2+)-dependent dimerization occurs predominantly by bridging an active site cysteine from either one of the active sites with one of the cysteines in the internal domain (b'). The dimer incorporates two atoms of Zn(2+) and exhibits 50% of the isomerase activity of PDI. At longer times and higher PDI concentrations, the dimer forms oligomers and aggregates of high molecular weight (>600 kDa). Because of a very high concentration of PDI in the ER, its interaction with divalent ions could play a role in regulating the effective concentration of these metal ions, protecting against metal toxicity, or affecting the activity of other (ER) proteins that use Zn(2+) as a cofactor.     

Heparan sulfates isolated from adult neural progenitor cells can direct phenotypic maturation

Multipotent progenitor stem cells that generate both neurons and glia are components of the hippocampus, subventricular zone and olfactory system of adult mammalian nervous system. The lineage choices any stem cell makes are known to be greatly dependent on the constitution of the extracellular matrix to which they are exposed during their development. Here, the adult rat hippocampus was used as a source of cells for clonal culture in order to investigate the effects of the extracellular glycosaminoglycan heparan sulfate (HS). Neurospheres were readily generated from adult tissue and could be used as a source of cells for further experiments. HS species that promote the actions of fibroblast growth factor-2 (FGF2) for embryonic neural progenitors were found to inhibit the actions of this mitogen for adult progenitors. Only HS fractions that promoted the actions of FGF1 had mitogenic effects on these adult cells. The adult cells proved difficult to clone from single cells. However, when endogenous HS was purified from these cells and added back at high concentration to single cells, the clones were capable of generating plentiful neuronal and glial progeny. The adult hippocampal progenitor (AHP) HS is composed of 32 kDa chains bearing 3 sulfated domains. A proportion of primary osteoblast stem cells exposed to the hippocampal HS adopt neuronal phenotypes. Hence, there appears to be a combination of HS-binding extracellular molecules that predispose cells to particular lineages.     

First report of <Emphasis Type="Italic">Candida bracarensis</Emphasis> in Mexico: hydrolytic enzymes and antifungal susceptibility pattern

Candida bracarensis is an emerging cryptic species within the Candida glabrata clade. To date, little is known about its epidemiology, virulence, and antifungal susceptibility. This study documents the occurrence of C. bracarensis for the first time in Mexico and focuses on its in vitro production of hydrolytic enzymes, as well as antifungal susceptibility to echinocandins. This strain was isolated from a vaginal swab of a female with vulvovaginal candidosis; exhibited a very strong activity of aspartyl proteinase, phospholipase, and hemolysin; and was susceptible to caspofungin, anidulafungin, and micafungin (MIC?=?0.031 μg/mL). Data obtained could contribute to the knowledge of the epidemiology and virulence attributes of this yeast as a fungal opportunistic human pathogen.