Re-engineering ketoacyl synthase specificity.
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3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Biomedical subjects
K Seshadri
Publications and source records attributed to K Seshadri.
Catalytic significance of the specificity of divalent cations as KS* and kcat* cofactors for secreted phospholipase A2.
Calcium is required for the substrate binding and for the chemical step of the interfacial catalytic turnover cycle of pancreatic phospholipase A2 (PLA2), but not for the binding of the enzyme to the interface. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate binding and kcat* for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of dimyristoylphosphatidylmethanol (DMPM) vesicles is characterized as an equilibrium dissociation constant for the interfacial binary (E*C) and ternary (E*CL) complexes of PLA2 and substrate mimics (L). Of the cations(II) that promote the binding of a mimic to the enzyme at the interface (E*), only a subgroup supports the chemical step. For example, Cd, Zn, and Cu form ternary E*CL complexes with kcat* of <1 s-1, compared to the rate of >100 s-1 with Ca, Fe, Mn, Co, and Ni. Oxygen exchange from H218O to the products of hydrolysis of DMPM incorporates one 18O in myristate. Incorporation of the first and second 18O occurs during the incubation of both the products of hydrolysis in H218O with PLA2 and Ca, but not with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of E*C and E*CL, suggest that the changes are mainly due to catalytic His-48, and possibly Tyr-52 and Tyr-73, and are different with Ca as opposed to Zn. These results and simulations suggest considerable plasticity in the calcium binding and catalytic site environment. It is proposed that the higher ground state stability of the E*CS complex with the inhibitory cations increases the effective activation energy. For the chemical step, calcium coordinated with a nucleophilic water and the ester carbonyl oxygen facilitates the near-attack geometry in the E*CaS, and the His-48.Asp-99 pair acts as a proton acceptor. As a prelude to establishing the catalytic mechanism, factors controlling the energetically demanding transition state are also discussed.
Animals↗
Architecture of beta-barrel membrane proteins: analysis of trimeric porins.
We have analyzed the known three-dimensional structures of trimeric porins from bacterial outer membranes. The distribution of surface-exposed residues in a direction perpendicular to the membrane is similar to that in helical membrane proteins, with aliphatic residues concentrated in the central 20 A of the bilayer. Outside these residues is a layer of aromatic residues, followed by polar and charged residues. Residues in the trimer interface are more conserved than residues not in the interface. By comparing the interface and noninterface residues, an interface preference scale has been derived that may be used as a basis for predicting interface surfaces in monomer models.
Bacterial Proteins↗
Structure of a glycoconjugate in solution and in complex with an antibody Fv fragment.
By use of heteronuclear (13C, 1H) NMR methods, the three-dimensional structure and dynamics of the glycoconjugate estrone-3-glucuronide (E3G) uniformly 13C enriched in the glucuronic acid moiety has been probed both in free solution and in association with an anti-E3G antibody single-chain Fv fragment. The glycan is found to exist in multiple conformations in free solution, with particularly large torsional fluctuations about the glycosidic linkage psi. Resonance assignments and distance restraints for the glycoconjugate in the bound state were obtained from heteronuclear proton-carbon-carbon-proton-COSY and isotope-edited NOESY techniques, respectively. Quantitation of the NOE data with a full-relaxation matrix approach showed that the antibody selects a conformation from the solution repertoire which does not correspond with either of the two lowest energy conformations of the free glycan, and the internal energy of the glycan in the bound state is estimated to be at most approximately 15 kcal/mol higher than the global minimum energy conformation. The glucuronide moiety undergoes a stacking interaction with an aromatic ring in the binding site, and both ring-current shifts and nuclear Overhauser effects computed from the predicted bound-state conformation are in good agreement with experiment. The bound-state conformation is also in good agreement with preliminary x-ray data on a related complex.
Carbohydrate Conformation↗
Structural and conformational analysis of glycan moieties in situ on isotopically 13C, 15N-enriched recombinant human chorionic gonadotropin.
The conformational properties in solution of the glycans on the alpha subunit of recombinant human chorionic gonadotropin are described, using high-resolution multinuclear NMR studies on uniformly 13C, 15N-enriched recombinant glycoprotein expressed in CHO cells. The glycan important for full biological activity of hCG, namely, that at Asn 52, appears to extend into solution both in the isolated alpha subunit and in complex with the beta subunit. The disposition of this glycan with respect to the protein backbone suggests that glycosylation maintains full biological activity of hCG either by interacting with a lectin-like region of the hCG receptor or by reducing the affinity of the hormone for the hCG receptor and preventing its down-regulation.
Carbohydrate Conformation↗
Interaction of substrate uridyl 3',5'-adenosine with ribonuclease A: a molecular dynamics study.
A wealth of information available from x-ray crystallographic structures of enzyme-ligand complexes makes it possible to study interactions at the molecular level. However, further investigation is needed when i) the binding of the natural substrate must be characterized, because ligands in the stable enzyme-ligand complexes are generally inhibitors or the analogs of substrate and transition state, and when ii) ligand binding is in part poorly characterized. We have investigated these aspects in the binding of substrate uridyl 3',5'-adenosine (UpA) to ribonuclease A (RNase A). Based on the systematically docked RNase A-UpA complex resulting from our previous study, we have undertaken a molecular dynamics simulation of the complex with solvent molecules. The molecular dynamics trajectories of this complex are analyzed to provide structural explanations for varied experimental observations on the ligand binding at the B2 subsite of ribonuclease A. The present study suggests that B2 subsite stabilization can be effected by different active site groups, depending on the substrate conformation. Thus when adenosine ribose pucker is O4'-endo, Gln69 and Glu111 form hydrogen-bonding contacts with adenine base, and when it is C2'-endo, Asn71 is the only amino acid residue in direct contact with this base. The latter observation is in support of previous mutagenesis and kinetics studies. Possible roles for the solvent molecules in the binding subsites are described. Furthermore, the substrate conformation is also examined along the simulation pathway to see if any conformer has the properties of a transition state. This study has also helped us to recognize that small but concerted changes in the conformation of the substrate can result in substrate geometry favorable for 2',3' cyclization. The identified geometry is suitable for intraligand proton transfer between 2'-hydroxyl and phosphate oxygen atom. The possibility of intraligand proton transfer as suggested previously and the mode of transfer before the formation of cyclic intermediate during transphosphorylation are discussed.
Animals↗
Characterization of substrate UpA binding to RNase A--computer modelling and energetics approach.
In the past two decades RNase A has been the focus of diverse investigations in order to understand the nature of substrate binding and to know the mechanism of enzyme action. Although this system is reasonably well characterized from the view point of some of the binding sites, the details of interactions in the second base binding (B2) site is insufficient. Further, the nature of ligand-protein interaction is elucidated generally by studies on RNase A-substrate analog complexes (mainly with the help of X-ray crystallography). Hence, the details of interactions at atomic level arising due to substrates are inferred indirectly. In the present paper, the dinucleotide substrate UpA is fitted into the active site of RNase A. Several possible substrate conformations are investigated and the binding modes have been selected based on Contact Criteria. Thus identified RNase A-UpA complexes are energy minimized in coordinate space and are analysed in terms of conformations, energetics and interactions. The best possible ligand conformations for binding to RNase A are identified by experimentally known interactions and by the energetics. Upon binding of UpA to RNase A, the changes associated with protein back bone, side chains in general and at the binding sites in particular are described. Further, the detailed interactions between UpA and RNase A are characterized in terms of hydrogen bonds and energetics. An extensive study has helped in interpreting the diverse results obtained from a number of experiments and also in evaluating the extent of changes the protein and the substrate undergo in order to maximize their interactions.
Animals↗
Are referrals to developmental pediatricians appropriate? Our experience over a 15-year period.
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Adolescent↗
Computer modelling studies of ribonuclease A-pyrimidine nucleotide complexes.
Different modes of binding of pyrimidine monophosphates 2'-UMP, 3'-UMP, 2'-CMP and 3'-CMP to ribonuclease (RNase) A are studied by energy minimization in torsion angle and subsequently in Cartesian coordinate space. The results are analysed in the light of primary binding sites. The hydrogen bonding pattern brings out roles for amino acids such as Asn44 and Ser123 apart from the well known active site residues viz., His12,Lys41,Thr45 and His119. Amino acid segments 43-45 and 119-121 seem to be guiding the ligand binding by forming a pocket. Many of the active site charged residues display considerable movement upon nucleotide binding.
Animals↗
Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates.
2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP,3'-UMP, A greater than p, G greater than p, U greater than p, C greater than p, GpA greater than p and UpA greater than p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.
Catalysis↗
18q- chromosomal abnormality in a phenotypically normal 2 1/2-year-old male with autism.
The authors report the case of a 2 1/2-year-old male with autism, with an 18q- chromosomal abnormality in the absence of phenotypical features of the 18q- syndrome. It is suggested that clinicians evaluating children with autistic disorders consider obtaining chromosome analysis, even in the absence of phenotypical abnormalities.
Autistic Disorder↗