Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it ...is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 Å resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting β-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge–charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.
Our aim was to characterize glomerular monocytes (Mo) infiltration and to correlate them with peripheral circulating Mo subsets and severity of lupus nephritis (LN). Methods. We evaluated 48 LN ...biopsy samples from a referral hospital. Recognition of Mo cells was done using microscopic view and immunohistochemistry stain with CD14 and CD16. Based on the number of cells, we classified LN samples as low degree of diffuse infiltration (<5 cells) and high degree of diffuse infiltration (≥5 cells). Immunophenotyping of peripheral Mo subsets was done using flow cytometry. Results. Mean age was 34.0±11.7 years and the mean SLEDAI was 17.5±6.9. The most common SLE manifestations were proteinuria (91%) and hypocomplementemia (75%). Severe LN was found in 70% of patients (Class III, 27%; Class IV, 43%). Severe LN patients and patients with higher grade of CD16+ infiltration had lower levels of nonclassical (CD14+CD16++) Mo in peripheral blood. Conclusions. Our results might suggest that those patients with more severe forms of LN had a higher grade of CD14+CD16+ infiltration and lower peripheral levels of nonclassical (CD14+CD16++) Mo and might reflect a recruitment process in renal tissues. However, given the small sample, our results must be interpreted carefully.
An integrated and sustainable fermentation process was developed which enabled both the revalorization of two regional agro-industrial discards as well as by-product reuse. Carrot and brewer's yeast, ...which are commonly used for animal feed, were processed to obtain 77.5 L of ethanol, 450 kg of solid waste called bagasse, 970 L of liquid effluent called vinasse, and 39.8 kg CO
2
per each ton of discarded carrot. Results showed that the obtained bagasse was suitable for feeding 55 animals (calfs). The dilution of vinasse with fresh water (1:5) satisfied the requirements necessary to be used as beverage for the same number of animals, leaving a remnant which could be newly diluted (1:5) and used to irrigate a 0.025-ha carrot crop, the land dimension required to grow 1 ton of carrot.
Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, ...a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
Thermophilic endo-1,3(4)-beta glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and ...contain two protein molecules in the asymmetric unit with a solvent content of 51.75 %. Diffraction data were collected to a resolution of 1.95A and resulted in a dataset with an overall R(merge) of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.
The enzyme β-xylosidase from Trichoderma reesei, a member of glycosil hydrolase family 3 (GH3), is a glycoside hydrolase which acts at the glycosidic linkages of 1,4-β-xylooligosaccharides and that ...also exhibits α-l-arabinofuranosidase activity on 4-nitrophenyl α-l-arabinofuranoside. In this work, we show that the enzyme forms monomers in solution and derive the low-resolution molecular envelope of the β-xylosidase from small-angle X-ray scattering (SAXS) data using the ab initio simulated annealing algorithm. The radius of gyration and the maximum dimension of the β-xylosidase are 30.3 ± 0.2 and 90 ± 5 Å, respectively. In contrast to the fold of the only two structurally characterized members of GH3, the barley β-d-glucan exohydrolase and β-hexosaminidase from Vibrio cholerae, which have respectively two or one distinct domains, the shape of the β-xylosidase indicates the presence of three distinct structural modules. Domain recognition algorithms were used to show that the C-terminal part of the amino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs demonstrate that this additional domain adopts a predominantly β conformation.
The
R
dx
A
oxygen‐insensitive nitroreductase of the human gastric pathogen
H
elicobacter pylori
is responsible for the susceptibility of this organism to the redox active prodrug metronidazole ...2‐(2‐methyl‐5‐nitro‐1
H
‐imidazol‐1‐yl)ethanol. Loss‐of‐function mutations in
rdxA
are primarily responsible for resistance to this therapeutic.
R
dx
A
exhibits potent
NADPH
oxidase activity under aerobic conditions and metronidazole reductase activity under strictly anaerobic conditions. In the present study, we report the crystal structure of
R
dx
A
, which is a homodimer exhibiting domain swapping and containing two molecules of
FMN
bound at the dimer interface. We have found a gap between the side chain of
T
yr47 and the isoalloxazine ring of
FMN
that appears to be appropriate for substrate binding. The structure does not include residues 97–128, which correspond to a locally unstable part of the
NTR
from
Escherichia coli
, and might be involved in cofactor binding. Comparison of
H
. pylori
R
dx
A
with other oxidoreductases of known structure suggests that
R
dx
A
may belong to a new subgroup of oxidoreductases in which a cysteine side chain close to the
FMN
cofactor could be involved in the reductive activity. In this respect, the mutation of C159 to
A
or
S
(
C
159
A
/
S
) has resulted in a loss of metronidazole reductase activity but not
NADPH
oxidase activity. The
R
dx
A
structure enables the interpretation of the many loss‐of‐function mutations described previously, including those affecting
C
159, a residue whose interaction with
FMN
is required for the nitroreduction of metronidazole. The present studies provide unique insights into the redox behaviour of the flavin in this key enzyme for metronidazole activation, including a potential use in gene therapy.
Database
Structural data have been deposited in the Protein Data Bank under accession number
3QDL
.
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