The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane. The channel structure must differ from that of the porintype open pore because the protein facilitates the exit of antibiotics but not the entry. For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-Å resolution. The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring ␤-barrel and the cavity-forming ␣-barrel. OprM anchors the outer membrane by two modes of membrane insertions. One is via the covalently attached NH 2 -terminal fatty acids and the other is the ␤-barrel structure consensus on the outer membrane-spanning proteins. The ␤-barrel had a pore opening with a diameter of about 6 -8 Å, which is not large enough to accommodate the exit of any antibiotics. The periplasmic ␣-barrel was about 100 Å long formed mainly by a bundle of ␣-helices that formed a solvent-filled cavity of about 25,000 Å 3 . The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule. The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics. However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends. The crystal structure offers possible mechanisms of pore opening and pump assembly.

The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the xray crystallographic structure of MexA at a resolution of 2.40 Å. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod ␣-helical domain was globular and constructed by a cluster of eight short ␤-sheets. The third domain located distal to the ␣-helical rod was globular and composed of seven short ␤-sheets and one short ␣-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic ␣-barrel domain of OprM via the ␣-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

A pair of tropinone reductases (TRs) share 64% of the same amino acid residues and belong to the short-chain dehydrogenase͞reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups with different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4-and 2.3-Å resolutions. The overall folding of the two enzymes was almost identical. The conservation was not confined within the core domains that are conserved within the protein family but extended outside the core domain where each family member has its characteristic structure. The binding sites for the cofactor and the positions of the active site residues were well conserved between the two TRs. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes. A modeling study indicated that these charged residues play a major role in controlling the binding orientation of tropinone within the substrate binding site, thereby determining the stereospecificity of the reaction product. The results obtained herein raise the possibility that in certain cases different stereospecificities can be acquired in enzymes by changing a few amino acid residues within substrate binding sites.

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From October 1988 to January 1992, nine isolates of Pseudomonas aeruginosa carrying transferable plasmids encoding imipenem-hydrolyzing beta-lactamase (pI = c. 9.5) were recovered from nine different patients in a neurosurgical ward of a hospital in Japan. The beta-lactamase activities of the sonicated extracts from the transconjugants were inhibited by EDTA and this was partially reversible by the addition of zinc cation. The substrate specificity and pI of the beta-lactamase were similar to those of the metallo beta-lactamases from P. aeruginosa and Serratia marcescens TN9106. All strains were resistant to imipenem, carbenicillin and antipseudomonal cephems including ceftazidime, cefsulodin, cefpirome, while four and five strains were susceptible to piperacillin and aztreonam, respectively. Both low level imipenem resistance and high level cephem resistance were co-transferred with the production of metallo beta-lactamase, while resistance to piperacillin, aztreonam, and high level imipenem-resistance were not selected. Production of chromosomal cephalosporinase in piperacillin resistant strains was derepressed, and production of outer membrane protein of D2 was diminished in highly imipenem resistant strains. Six strains were isolated in 1991, and the amounts of antipseudomonal agents, especially imipenem, used in the neurosurgical ward increased markedly in this year. Only three of the nine isolates had the same serotype, pyocin type and phage type. Our results suggest that the repeated isolation of imipenem and cephem-resistant P. aeruginosa producing metallo beta-lactamase was related to the high usage of antipseudomonal beta-lactam antibiotics such as imipenem, and was exacerbated by the dissemination of a plasmid.