RESUMEN

Uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase (MurB), the second enzyme in the peptidoglycan synthetic pathway of Escherichia coli, has been crystallized in two previously unreported forms, one orthorhombic and the other monoclinic. MurB (molecular mass 38 kDa) crystallizes in a range of conditions that utilize polyethylene glycol fractions as precipitants, and crystals can be grown with or without the enzyme's substrate, uridine 5'-diphospho-N-acetylenolpyruvylglucosamine. X-ray diffraction from crystals of the orthorhombic form extends to 2 A resolution and shows the symmetry and systematic absences of space group P2(1)2(1)2(1). These crystals show significant variations in cell dimensions at room temperature and at 100 K. A crystal used to collect a 2.0 A resolution data set at a synchrotron source showed cell dimensions at ca 100 K of a = 51.0, b = 79.3 and c = 87.1 A, indicating one molecule peroasymmetric unit. The monoclinic crystals scatter X-rays to 3.0 A resolution consistent with space group P2(1), unit-cell dimensions (ca 100 K) a = 50.7, b = 92.4, c = 85.5 A, and beta = 104 degrees, and two molecules per asymmetric unit. Mercury derivatives have been prepared with both orthorhombic and monoclinic forms, and efforts are underway to exploit these derivatives to determine the structure of this protein.

RESUMEN

The crystallographic structures of the ternary complexes of human alpha-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt very similar bound conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.

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The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein required for the assembly of plasma very low density lipoproteins in the liver and chylomicrons in the intestine. Bovine MTP was purified by a modification of a previously published procedure and crystals of MTP were grown reproducibly with polyethylene glycol as a precipitant at pH 7.0. MTP crystals, which diffract to Bragg spacings of better than 3.2 A, have the symmetry of space group P2(1)2(1)2(1) with refined lattice constants of a = 88.7, b = 100.9 and c = 201.1 A, with one heterodimer per asymmetric unit.

RESUMEN

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

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Investigated the association between various depression assessment methods in 38 adults with mild or moderate mental retardation, half of whom had relatively high and the other half had relatively low depression screening scores. Measures included a standard psychiatric interview (Diagnostic Interview for Children and Adolescents), an informant rating scale (Reiss Screen for Maladaptive Behavior), and a self-report measure (Self-Report Depression Questionnaire). Association between measures was generally low, yielding discordant classification results. Potential reasons for these discrepancies were offered, and implications for clinical and research assessment of mood disorders in mental retardation were discussed.

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An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediated TEM-1 beta-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibility to extended-spectrum beta-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, but it decreased the MICs of the penicillins. The TEM-1-producing plasmid was transferred to a more susceptible S. marcescens strain that produced a well-characterized inducible chromosomal beta-lactamase. The MICs of the ESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin which were good substrates for the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these beta-lactam antibiotics, at least modest increases in chromosomal beta-lactamase activity were observed. When organisms were grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. The increases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combination of low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. When clavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a chromosomal beta-lactamase, much higher levels of chromosomal beta-lactamase activity were present than were observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were available for induction as a result of inhibition of the TEM enzyme by clavulanate.

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By site-directed mutagenesis, TEM-1 beta-lactamase was altered to contain single amino acid changes of E104K, R164S, and E240K, in addition to double changes of E104K/R164S or R164S/E240K and the triple change of E104K/R164S/E240K. Hydrolysis rates for cephaloridine and benzylpenicillin were lowered at least 1 order of magnitude for all enzymes containing R164S substitutions. All mutant enzymes exhibited increased kcat values for beta-lactam antibiotics containing an aminothiazole oxime side chain. Hydrolysis of ceftazidime was most affected, with kcat values increased 3-4 orders of magnitude in all enzymes with the substituted R164S moiety. Km values decreased for all substrates except ceftazidime in the enzymes with multiple mutations. Aztreonam was most affected, with Km values lowered 23-56-fold in the enzymes bearing multiple mutations. When the crystal structures of aztreonam and related monobactams were studied and projected into an active-site model of the PC1 beta-lactamase, it became apparent that the two lysine residues might serve equivalent roles by interacting with the carboxylate of the aminothiazole oxime side chain. Hydrogen-bonding interactions involving the oxime and N7 of the lysine, particularly Lys-104, may also be important in some antibiotics. Ser-164 apparently serves an indirect role, since it is somewhat distant from the active-site cleft.

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Tigemonam and temocillin, but not aztreonam, bound to penicillin-binding proteins (PBPs) 1a and 3 of Escherichia coli with apparent improved affinity when challenged with benzylpenicillin at lowered temperatures. Half times for deacylation of the tigemonam-PBP complexes were shorter than were those of the corresponding aztreonam-PBP complexes. The implications of the routine testing of PBP affinities are discussed.

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