68 research outputs found
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Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3-ketosteroid to its conjugated Δ4-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI
Ground State Destabilization from a Positioned General Base in the Ketosteroid Isomerase Active Site
Decoding the structural bases of D76N ß2-microglobulin high amyloidogenicity through crystallography and Asn-Scan mutagenesis
D76N is the first natural variant of human \u3b2-2 microglobulin (\u3b22m) so far identified. Contrary to the wt protein, this mutant readily forms amyloid fibres in physiological conditions, leading to a systemic and severe amyloidosis. Although the Asp76Asn mutant has been extensively characterized, the molecular bases of its instability and aggregation propensity remain elu- sive. In this work all Asp residues of human \u3b22m were individually substituted to Asn; D-to- N mutants (D34N, D38N, D53N, D59N, D96N and D98N) were characterised in terms of thermodynamic stability and aggregation propensity. Moreover, crystal structures of the D38N, D53N, D59N and D98N variants were solved at high-resolution (1.24\u20131.70 \uc5). Despite showing some significant variations in their thermal stabilities, none showed the dramatic drop in melting temperature (relative to the wt protein) as observed for the patho- genic mutant. Consistently, none of the variants here described displayed any increase in aggregation propensity under the experimental conditions tested. The crystal structures confirmed that D-to-N mutations are generally well tolerated, and lead only to minor reorga- nization of the side chains in close proximity of the mutated residue. D38N is the only excep- tion, where backbone readjustments and a redistribution of the surface electrostatic charges are observed. Overall, our results suggest that neither removing negative charges at sites 34, 38, 53, 59, 96 and 98, nor the difference in \u3b22m pI, are the cause of the aggres- sive phenotype observed in D76N. We propose that the dramatic effects of the D76N natu- ral mutation must be linked to effects related to the crucial location of this residue within the \u3b22m fold
Lethal mutations in the isoprenoid pathway of salmonella enterica
Journal ArticleEssential isoprenoid compounds are synthesized using the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of Salmonella enterica serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway
Forced Decryption as Equilibrium—Why It’s Constitutional and How Riley Matters
This Essay considers whether the government can force a person to decrypt his computer. The only courts to consider the issue limited their analyses to rote application of predigital doctrine and dicta. This is a mistake; courts should instead aim to maintain the ex ante equilibrium of privacy and government power. This approach—seeking equilibrium—was just endorsed by the Supreme Court in Riley v. California, a recent Fourth Amendment case. Yet Riley’s rationale also extends to the Fifth Amendment’s Self-Incrimination Clause, and maintaining equilibrium there requires permitting forced decryption. Because current doctrine can be interpreted as allowing forced decryption, courts should adopt that interpretation
An Asp to Asn mutation is a toxic trigger in beta-2 microglobulin: structure and biophysics
Beta-2 microglobulin (β2m) is part of the Major Histocompatibility Complex Class I (MHC I) and when monomeric becomes an aggregation prone protein that is responsible for a human disorder known as dialysis-related amyloidosis. In 2012 Valleix et al. described a new familial systemic amyloidosis: an unreported β2m mutant (D76N) is the etiological agent of such disease. Main symptoms were chronic diarrhea, loss of weight and polyneuropathy: large amyloid deposits were found in internal organs. From the biophysical point of view, the D76N β2m is much less stable and more amyloidogenic than wt β2m; however, its crystal structure reveals very minor conformational changes compared with the wt protei
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Engineered MATE multidrug transporters reveal two functionally distinct ion-coupling pathways in NorM from Vibrio cholerae.
Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H+ or Na+ gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H+ and Na+ translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H+-coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae
Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole
A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K (a) models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K (a) unit, suggesting a bond shortening of ˜0.02 Å/p K (a) unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K (a) unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K (a) unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution
Geodetic Graphs: Experiments and New Constructions
In 1962 Ore initiated the study of geodetic graphs. A graph is called
geodetic if the shortest path between every pair of vertices is unique. In the
subsequent years a wide range of papers appeared investigating their peculiar
properties. Yet, a complete classification of geodetic graphs is out of reach.
In this work we present a program enumerating all geodetic graphs of a given
size. Using our program, we succeed to find all geodetic graphs with up to 25
vertices and all regular geodetic graphs with up to 32 vertices. This leads to
the discovery of two new infinite families of geodetic graphs
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