Protonation states of active-site lysines of penicillin-binding protein 6 from Escherichia coli and the mechanistic implications.

The protonation states of the two active-site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S-aminoethylated cysteine (γ-thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd-carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free-base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pKa values were compared with the pKa values calculated computationally, using molecular-dynamics simulations and a thermodynamic cycle. Study of the γ-thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half-reactions.

Characterization of the dimerization interface of membrane type 4 (MT4)-matrix metalloproteinase.

MT4-MMP (MMP17) belongs to a unique subset of membrane type-matrix metalloproteinases that are anchored to the cell surface via a glycosylphosphatidylinositol moiety. However, little is known about its biochemical properties. Here, we report that MT4-MMP is displayed on the cell surface as a mixed population of monomeric, dimeric, and oligomeric forms. Sucrose gradient fractionation demonstrated that these forms of MT4-MMP are all present in lipid rafts. Mutational and computational analyses revealed that Cys(564), which is present within the stem region, mediates MT4-MMP homodimerization by forming a disulfide bond. Substitution of Cys(564) results in a more rapid MT4-MMP turnover, when compared with the wild-type enzyme, consistent with a role for dimerization in protein stability. Expression of MT4-MMP in Madin-Darby canine kidney cells enhanced cell migration and invasion of Matrigel, a process that requires catalytic activity. However, a serine substitution at Cys(564) did not reduce MT4-MMP-stimulated cell invasion of Matrigel suggesting that homodimerization is not required for this process. Deglycosylation studies showed that MT4-MMP is modified by N-glycosylation. Moreover, inhibition of N-glycosylation by tunicamycin diminished the extent of MT4-MMP dimerization suggesting that N-glycans may confer stability to the dimeric form. Taken together, the data presented here provide a new insight into the characteristics of MT4-MMP and highlight the common and distinct properties of the glycosylphosphatidylinositol-anchored membrane type-matrix metalloproteinases.

Lysine Nzeta-decarboxylation switch and activation of the beta-lactam sensor domain of BlaR1 protein of methicillin-resistant Staphylococcus aureus.

The integral membrane protein BlaR1 of methicillin-resistant Staphylococcus aureus senses the presence of ß-lactam antibiotics in the milieu and transduces the information to the cytoplasm, where the biochemical events that unleash induction of antibiotic resistance mechanisms take place. We report herein by two-dimensional and three-dimensional NMR experiments of the sensor domain of BlaR1 in solution and by determination of an x-ray structure for the apo protein that Lys-392 of the antibiotic-binding site is posttranslationally modified by N(ζ)-carboxylation. Additional crystallographic and NMR data reveal that on acylation of Ser-389 by antibiotics, Lys-392 experiences N(ζ)-decarboxylation. This unique process, termed the lysine N(ζ)-decarboxylation switch, arrests the sensor domain in the activated ("on") state, necessary for signal transduction and all the subsequent biochemical processes. We present structural information on how this receptor activation process takes place, imparting longevity to the antibiotic-receptor complex that is needed for the induction of the antibiotic-resistant phenotype in methicillin-resistant S. aureus.

A computational evaluation of the mechanism of penicillin-binding protein-catalyzed cross-linking of the bacterial cell wall.

Penicillin-binding protein 1b (PBP 1b) of the gram-positive bacterium Streptococcus pneumoniae catalyzes the cross-linking of adjacent peptidoglycan strands, as a critical event in the biosynthesis of its cell wall. This enzyme is representative of the biosynthetic PBP structures of the ß-lactam-recognizing enzyme superfamily and is the target of the ß-lactam antibiotics. In the cross-linking reaction, the amide between the -D-Ala-D-Ala dipeptide at the terminus of a peptide stem acts as an acyl donor toward the ε-amino group of a lysine found on an adjacent stem. The mechanism of this transpeptidation was evaluated using explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics calculations. Sequential acyl transfer occurs to, and then from, the active site serine. The resulting cross-link is predicted to have a cis-amide configuration. The ensuing and energetically favorable cis- to trans-amide isomerization, within the active site, may represent the key event driving product release to complete enzymatic turnover.

QM/MM Studies of the Matrix Metalloproteinase 2 (MMP2) Inhibition Mechanism of (S)-SB-3CT and its Oxirane Analogue.

SB-3CT, (4-phenoxyphenylsulfonyl)methylthiirane, is a potent, mechanism-based inhibitor of the gelatinase sub-class of the matrix metalloproteinase (MMP) family of zinc proteases. The gelatinase MMPs are unusual in that there are several examples where both enantiomers of a racemic inhibitor have comparable inhibitory abilities. SB-3CT is one such example. Here, the inhibition mechanism of the MMP2 gelatinase by the (S)-SB-3CT enantiomer and its oxirane analogue is examined computationally, and compared to the mechanism of (R)-SB-3CT. Inhibition of MMP2 by (R)-SB-3CT was shown previously to involve enzyme-catalyzed C-H deprotonation adjacent to the sulfone, with concomitant opening by ß-elimination of the sulfur of the three-membered thiirane ring. Similarly to the R enantiomer, (S)-SB-3CT was docked into the active site of MMP2, followed by molecular dynamics simulation to prepare the complex for combined quantum mechanics and molecular mechanics (QM/MM) calculations. QM/MM calculations with B3LYP/6-311+G(d,p) for the QM part (46 atoms) and the AMBER force field for the MM part were used to compare the reaction of (S)-SB-3CT and its oxirane analogue in the active site of MMP2 (9208 atoms). These calculations show that the barrier for the proton abstraction coupled ring opening reaction of (S)-SB-3CT in the MMP2 active site is 4.4 kcal/mol lower than its oxirane analogue, and the ring opening reaction energy of (S)-SB-3CT is only 1.6 kcal/mol less exothermic than its oxirane analogue. Calculations also show that the protonation of the ring-opened products by water is thermodynamically much more favorable for the alkoxide obtained from the oxirane, than for the thiolate obtained from the thiirane. In contrast to (R)-SB-3CT and the R-oxirane analogue, the double bonds of the ring-opened products of (S)-SB-3CT and its S-oxirane analogue have the cis-configuration. Vibrational frequency and intrinsic reaction path calculations on a reduced size QM/MM model (2747 atoms) provide additional insight into the mechanism. These calculations yield 5.9 and 6.7 for the deuterium kinetic isotope effect for C-H bond cleavage in the transition state for the R and S enantiomers of SB-3CT, in good agreement with the experimental results.

Elucidation of the structure of the membrane anchor of penicillin-binding protein 5 of Escherichia coli.

Penicillin-binding protein 5 (PBP 5) of Escherichia coli is a membrane-bound cell wall dd-carboxypeptidase, localized in the outer leaflet of the cytosolic membrane of this Gram-negative bacterium. Not only is it the most abundant PBP of E. coli, but it is as well a target for penicillins and is the most studied of the PBP enzymes. PBP 5, as a representative peripheral membrane protein, is anchored to the cytoplasmic membrane by the 21 amino acids of its C-terminus. Although the importance of this terminus as a membrane anchor is well recognized, the structure of this anchor was previously unknown. Using natural isotope abundance NMR, the structure of the PBP 5 anchor peptide within a micelle was determined. The structure conforms to a helix-bend-helix-turn-helix motif and reveals that the anchor enters the membrane so as to form an amphiphilic structure within the interface of the hydrophilic/hydrophobic boundary regions near the lipid head groups. The bend and the turn within the motif allow the C-terminus to exit from the same side of the membrane that is penetrated. The PBP anchor sequences represent extraordinary diversity, encompassing both N-terminal and C-terminal anchoring domains. This study establishes a surface adherence mechanism for the PBP 5 C-terminus anchor peptide, as the structural basis for further study toward understanding the role of these domains in selecting membrane environments and in the assembly of the multienzyme hyperstructures of bacterial cell wall biosynthesis.

Matrix metalloproteinase 2 (MMP2) inhibition: DFT and QM/MM studies of the deprotonation-initialized ring-opening reaction of the sulfoxide analogue of SB-3CT.

(4-Phenoxyphenylsulfonyl)methylthiirane (SB-3CT) is the selective inhibitor of matrix metalloproteinase 2 (MMP2). The inhibition mechanism of MMP2 by SB-3CT involves C-H deprotonation with concomitant opening of the three-membered heterocycle. In this study, the energetics of the deprotonation-induced ring-opening of (4-phenoxyphenylsulfinyl)methylthiirane, the sulfoxide analogue of SB-3CT, are examined computationally using DFT and QM/MM calculations. A model system, 2-(methylsulfinylmethyl)thiirane, is used to study the stereoelectronic and conformational effects of reaction barriers in methanol. For the model system in methanol solution (using the polarizable continuum model), the reaction barriers range from 17 to 23 kcal/mol with significant stereoelectronic effects. However, the lowest barriers of the (R,R) and (S,R) diastereomers are similar. Two diastereomers of the sulfoxide analogue of SB-3CT are studied in the active site of MMP2 by QM/MM methods with an accurate partial charge fitting procedure. The ring-opening reactions of these two diastereomers have similar reaction energetics. Both are exothermic from the reactant to the ring-opening product (thiolate). The protonation of the thiolate by a water molecule is endothermic in both cases. However, the deprotonation/ring-opening barriers in the MMP2 active site using QM/MM methods for the (R,R) and (S,R) inhibitions are quite different (23.3 and 28.5 kcal/mol, respectively). The TSs identified in QM/MM calculations were confirmed by vibrational frequency analysis and following the reaction path. The (R,R) diastereomer has a hydrogen bond between the sulfoxide oxygen and the backbone NH of Leu191, while the (S,R) has a hydrogen bond between the sulfoxide oxygen and a water molecule. The dissimilar strengths of these hydrogen bonds as well as minor differences in the TS structures contribute to the difference between the barriers. Compared to SB-3CT, both diastereomers of the sulfoxide analogue have higher reaction barriers and have less exothermic reaction energies. This agrees well with the experiments, where SB-3CT is a more effective inhibitor of MMP2 than its sulfoxide analogue.

Crystal structures of penicillin-binding protein 6 from Escherichia coli.

Penicillin-binding protein 6 (PBP6) is one of the two main DD-carboxypeptidases in Escherichia coli, which are implicated in maturation of bacterial cell wall and formation of cell shape. Here, we report the first X-ray crystal structures of PBP6, capturing its apo state (2.1 A), an acyl-enzyme intermediate with the antibiotic ampicillin (1.8 A), and for the first time for a PBP, a preacylation complex (a "Michaelis complex", determined at 1.8 A) with a peptidoglycan substrate fragment containing the full pentapeptide, NAM-(L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala). These structures illuminate the molecular interactions essential for ligand recognition and catalysis by DD-carboxypeptidases, and suggest a coupling of conformational flexibility of active site loops to the reaction coordinate. The substrate fragment complex structure, in particular, provides templates for models of cell wall recognition by PBPs, as well as substantiating evidence for the molecular mimicry by beta-lactam antibiotics of the peptidoglycan acyl-D-Ala-D-Ala moiety.

Active site ring-opening of a thiirane moiety and picomolar inhibition of gelatinases.

(+/-)-2-[(4-Phenoxyphenylsulfonyl)methyl]thiirane 1 is a potent and selective mechanism-based inhibitor of the gelatinase sub-class of the zinc-dependent matrix metalloproteinase family. Inhibitor 1 has excellent activity in in vivo models of gelatinase-dependent disease. We demonstrate that the mechanism of inhibition is a rate-limiting gelatinase-catalyzed thiolate generation via deprotonation adjacent to the thiirane, with concomitant thiirane opening. A corollary to this mechanism is the prediction that thiol-containing structures, related to thiirane-opened 1, will possess potent matrix metalloproteinase inhibitory activity. This prediction was validated by the synthesis of the product of this enzyme-catalyzed reaction on 1, which exhibited a remarkable K(i) of 530 pm against matrix metalloproteinase-2. Thiirane 1 acts as a caged thiol, unmasked selectively in the active sites of gelatinases. This mechanism is unprecedented in the substantial literature on inhibition of zinc-dependent hydrolases.

Matrix metalloproteinase 2 inhibition: combined quantum mechanics and molecular mechanics studies of the inhibition mechanism of (4-phenoxyphenylsulfonyl)methylthiirane and its oxirane analogue.

The inhibition mechanism of matrix metalloproteinase 2 (MMP2) by the selective inhibitor (4-phenoxyphenylsulfonyl)methylthiirane (SB-3CT) and its oxirane analogue is investigated computationally. The inhibition mechanism involves C-H deprotonation with concomitant opening of the three-membered heterocycle. SB-3CT was docked into the active site of MMP2, followed by molecular dynamics simulation to prepare the complex for combined quantum mechanics and molecular mechanics (QM/MM) calculations. QM/MM calculations with B3LYP/6-311+G(d,p) for the QM part and the AMBER force field for the MM part were used to examine the reaction of these two inhibitors in the active site of MMP2. The calculations show that the reaction barrier for transformation of SB-3CT is 1.6 kcal/mol lower than its oxirane analogue, and the ring-opening reaction energy of SB-3CT is 8.0 kcal/mol more exothermic than that of its oxirane analogue. Calculations also show that protonation of the ring-opened product by water is thermodynamically much more favorable for the alkoxide obtained from the oxirane than for the thiolate obtained from the thiirane. A six-step partial charge fitting procedure is introduced for the QM/MM calculations to update atomic partial charges of the quantum mechanics region and to ensure consistent electrostatic energies for reactants, transition states, and products.

Mechanistic basis for the emergence of catalytic competence against carbapenem antibiotics by the GES family of beta-lactamases.

A major mechanism of bacterial resistance to beta-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is the production of beta-lactamases. A handful of class A beta-lactamases have been discovered that have acquired the ability to turn over carbapenem antibiotics. This is a disconcerting development, as carbapenems are often considered last resort antibiotics in the treatment of difficult infections. The GES family of beta-lactamases constitutes a group of extended spectrum resistance enzymes that hydrolyze penicillins and cephalosporins avidly. A single amino acid substitution at position 170 has expanded the breadth of activity to include carbapenems. The basis for this expansion of activity is investigated in this first report of detailed steady-state and pre-steady-state kinetics of carbapenem hydrolysis, performed with a class A carbapenemase. Monitoring the turnover of imipenem (a carbapenem) by GES-1 (Gly-170) revealed the acylation step as rate-limiting. GES-2 (Asn-170) has an enhanced rate of acylation, compared with GES-1, and no longer has a single rate-determining step. Both the acylation and deacylation steps are of equal magnitude. GES-5 (Ser-170) exhibits an enhancement of the rate constant for acylation by a remarkable 5000-fold, whereby the enzyme acylation event is no longer rate-limiting. This carbapenemase exhibits k(cat)/K(m) of 3 x 10(5) m(-1)s(-1), which is sufficient for manifestation of resistance against imipenem.

Regiospecific syntheses of 6alpha-(1R-Hydroxyoctyl)penicillanic acid and 6beta-(1R-hydroxyoctyl)penicillanic acid as mechanistic probes of class D beta-lactamases.

The unique hydrophobic surface patches in class D beta-lactamases presented an opportunity for designing two compounds, 6alpha-(1R-hydroxyoctyl)penicillanic acid and 6beta-(1R-hydroxyoctyl)penicillanic acid, as mechanistic probes of these enzymes. In a sequence of three synthetic steps from benzhydryl 6,6-dibromopenicillanate, the targeted compounds were prepared in a stereospecific manner.

A potent gelatinase inhibitor with anti-tumor-invasive activity and its metabolic disposition.

Metastatic tumors lead to more than 90% fatality. Despite the importance of invasiveness of tumors to poor disease outcome, no anti-invasive compounds have been commercialized. We describe herein the synthesis and evaluation of 4-(4-(thiiranylmethylsulfonyl)phenoxy)-phenyl methanesulfonate (compound 2) as a potent and selective inhibitor of gelatinases (matrix metalloproteinases-2 and -9), two enzymes implicated in invasiveness of tumors. It was demonstrated that compound 2 significantly attenuated the invasiveness of human fibrosarcoma cells (HT1080). The metabolism of compound 2 involved hydroxylation at the alpha-methylene, which generates sulfinic acid, thiirane ring-opening, followed by methylation and oxidation, and cysteine conjugation of both the thiirane and phenyl rings.

Identification and role of the homodimerization interface of the glycosylphosphatidylinositol-anchored membrane type 6 matrix metalloproteinase (MMP25).

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Investigation of the mechanism of the cell wall DD-carboxypeptidase reaction of penicillin-binding protein 5 of Escherichia coli by quantum mechanics/molecular mechanics calculations.

Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem peptides of the cell wall peptidoglycan. The mechanism of PBP 5 catalysis of amide bond hydrolysis is initial acylation of an active site serine by the peptide substrate, followed by hydrolytic deacylation of this acyl-enzyme intermediate to complete the turnover. The microscopic events of both the acylation and deacylation half-reactions have not been studied. This absence is addressed here by the use of explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations. The potential-energy surface for the acylation reaction, based on MP2/6-31+G(d) calculations, reveals that Lys47 acts as the general base for proton abstraction from Ser44 in the serine acylation step. A discrete potential-energy minimum for the tetrahedral species is not found. The absence of such a minimum implies a conformational change in the transition state, concomitant with serine addition to the amide carbonyl, so as to enable the nitrogen atom of the scissile bond to accept the proton that is necessary for progression to the acyl-enzyme intermediate. Molecular dynamics simulations indicate that transiently protonated Lys47 is the proton donor in tetrahedral intermediate collapse to the acyl-enzyme species. Two pathways for this proton transfer are observed. One is the direct migration of a proton from Lys47. The second pathway is proton transfer via an intermediary water molecule. Although the energy barriers for the two pathways are similar, more conformers sample the latter pathway. The same water molecule that mediates the Lys47 proton transfer to the nitrogen of the departing D-Ala is well positioned, with respect to the Lys47 amine, to act as the hydrolytic water in the deacylation step. Deacylation occurs with the formation of a tetrahedral intermediate over a 24 kcal x mol(-1) barrier. This barrier is approximately 2 kcal x mol(-1) greater than the barrier (22 kcal x mol(-1)) for the formation of the tetrahedral species in acylation. The potential-energy surface for the collapse of the deacylation tetrahedral species gives a 24 kcal x mol(-1) higher energy species for the product, signifying that the complex would readily reorganize and pave the way for the expulsion of the product of the reaction from the active site and the regeneration of the catalyst. These computational data dovetail with the knowledge on the reaction from experimental approaches.

Aminoglycoside 2''-phosphotransferase type IIIa from Enterococcus.

Aminoglycoside 2''-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the treatment of serious enterococcal infections. This work reports on cloning, purification, and detailed mechanistic characterization of aminoglycoside 2''-phosphotransferase, known as type Ic enzyme. In an unexpected finding, the enzyme exhibits strong preference for guanosine triphosphate over adenosine triphosphate as the phosphate donor, a unique observation among all characterized aminoglycoside phosphotransferases. The enzyme phosphorylates only certain 4,6-disubstituted aminoglycosides exclusively at the 2''-hydroxyl with k(cat) values of 0.5-1.0 s(-1) and K(m) values in the nanomolar range for all substrates but kanamycin A. Based on this unique substrate profile, the enzyme is renamed aminoglycoside 2''-phosphotransferase type IIIa. Product and dead-end inhibition patterns indicated a random sequential Bi Bi mechanism. Both the solvent viscosity effect and determination of the rate constant for dissociation of guanosine triphosphate indicated that at pH 7.5 the release of guanosine triphosphate is rate-limiting. A computational model for the enzyme is presented that sheds light on the structural aspects of interest in this family of enzymes.

Conformational analyses of thiirane-based gelatinase inhibitors.

SB-3CT is a thiirane-containing inhibitor of the gelatinase class of matrix metalloprotease enzymes. In support of the mechanistic study of this inhibition, the conformational analyses of SB-3CT (and of two methyl-substituted derivatives) were undertaken using X-ray crystallography and molecular dynamics simulation.

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli.

Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.

Shared functional attributes between the mecA gene product of Staphylococcus sciuri and penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus.

The genome of Staphylococcus aureus is constantly in a state of flux, acquiring genes that enable the bacterium to maintain resistance in the face of antibiotic pressure. The acquisition of the mecA gene from an unknown origin imparted S. aureus with broad resistance to beta-lactam antibiotics, with the resultant strain designated as methicillin-resistant S. aureus (MRSA). Epidemiological and genetic evidence suggests that the gene encoding PBP 2a of MRSA might have originated from Staphylococcus sciuri, an animal pathogen, where it exists as a silent gene of unknown function. We synthesized, cloned, and expressed the mecA gene of S. sciuri in Escherichia coli, and the protein product was purified to homogeneity. Biochemical characterization and comparison of the protein to PBP 2a of S. aureus revealed them to be highly similar. These characteristics start with sequence similarity but extend to biochemical behavior in inhibition by beta-lactam antibiotics, to the existence of an allosteric site for binding of bacterial peptidoglycan, to the issues of the sheltered active site, and to the need for conformational change in making the active site accessible to the substrate and the inhibitors. Altogether, the evidence strongly argues that the kinship between the two proteins is deep-rooted on the basis of many biochemical attributes quantified in this study.

Design and synthesis of a structurally constrained aminoglycoside.

The synthesis of a constrained tricyclic aminoglycoside derivative is described. This constrained compound fixes the spatial orientation of two critical rings for the minimal motif for binding to biological macromolecules such as RNA and proteins. Methanolysis of neomycin B under acidic conditions produced the bicyclic neamine. Transient protection by the Cu2+ ion and regioselective introduction of protective groups led to intermediate 7, which was used for a key annulation reaction that introduced the tricyclic nucleus into the structural framework. A final hydrogenolysis step to remove the protective groups produced the desired target molecule. The efficient eight-step synthesis was accomplished in 8% overall yield.