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The heteroepitaxial growth of rutile and VO2 thin films and multilayers of rutile/VO2 on sapphire substrates is discussed. A metalorganic chemical vapor deposition technique was used to grow the epitaxial thin films on the substrates as Raman scattering and four-circle x-ray diffractometry characterized them. The thin films exhibited a high degree of orientation in both parallel and perpendicular placements to the growth plane.

Presents results from a study on the structure of grain boundaries and magnetic domains in the soft magnetic material nickel-iron compound. Experimental details; Nuclear and magnetic scattering profiles; Conclusions.

Presents a study that investigated Raman scattering and x-ray diffractometry of epitaxial TiO[sub2] and VO[sub2] thin films and multilayers on α-Al[sub2]O[sub3]. Analysis of x-ray diffraction results; Discussion on the adherence of single-layer films to Raman selection rules of TiO[sub2] and VO[sub2] single crystals; Details on growth conditions and film composition.

Inelastic neutron scattering spectra were measured on Ce metal at temperatures near the fcc (gamma) to bcc (delta) transition, and phonon DOS curves were obtained. A large difference in the phonon DOS of gamma-Ce and delta-Ce was found, and this difference accounts for a change in vibrational entropy of (0.51 0.05)kB atom-1 at the gamma-delta transition. To be consistent with the latent heat of the gamma-delta transition, this large change in vibrational entropy should be accompanied by a thermodynamically-significant change in electronic entropy of the opposite sign. [ABSTRACT FROM AUTHOR]

Some members of the class A β-lactamase family are capable of conferring resistance to the last resort antibiotics, carbapenems. A unique structural feature of these clinically important enzymes, collectively referred to as class A carbapenemases, is a disulfide bridge between invariant Cys 69 and Cys 238 residues. It was proposed that this conserved disulfide bridge is responsible for their carbapenemase activity, but this has not yet been validated. Here we show that disruption of the disulfide bridge in the GES-5 carbapenemase by the C69G substitution results in only minor decreases in the conferred levels of resistance to the carbapenem imipenem and other β-lactams. Kinetic and circular dichroism experiments with C69G-GES-5 demonstrate that this small drop in antibiotic resistance is due to a decline in the enzyme activity caused by a marginal loss of its thermal stability. The atomic resolution crystal structure of C69G-GES-5 shows that two domains of this disulfide bridge-deficient enzyme are held together by an intensive hydrogen-bonding network. As a result, the protein architecture and imipenem binding mode remain unchanged. In contrast, the corresponding hydrogen-bonding networks in NMCA, SFC-1, and SME-1 carbapenemases are less intensive, and as a consequence, disruption of the disulfide bridge in these enzymes destabilizes them, which causes arrest of bacterial growth. Our results demonstrate that the disulfide bridge is essential for stability but does not play a direct role in the carbapenemase activity of the GES family of β-lactamases. This would likely apply to all other class A carbapenemases given the high degree of their structural similarity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Production of β-lactamases of one of four molecular classes (A, B, C and D) is the major mechanism of bacterial resistance to β-lactams, the largest class of antibiotics, which have saved countless lives since their inception 70 years ago. Although several hundred efficient class D enzymes have been identified in Gram-negative pathogens over the last four decades, none have been reported in Gram-positive bacteria. Here we demonstrate that efficient class D β-lactamases capable of hydrolyzing a wide array of β-lactam substrates are widely disseminated in various species of environmental Gram-positive organisms. Class D enzymes of Gram-positive bacteria have a distinct structural architecture and employ a unique substrate-binding mode that is quite different from that of all currently known class A, C and D β-lactamases. These enzymes thus constitute a previously unknown reservoir of novel antibiotic-resistance enzymes.

Class D β-lactamases of Acinetobacter baumannii are enzymes of the utmost clinical importance, producing resistance to last resort carbapenem antibiotics. Although the OXA-51-like enzymes constitute the largest family of class D β-lactamases, they are poorly studied and their importance in conferring carbapenem resistance is controversial. We present the detailed microbiological, kinetic, and structural characterization of the eponymous OXA-51 β-lactamase. Kinetic studies show that OXA-51 has low catalytic efficiency for carbapenems, primarily due to the low affinity of the enzyme for these substrates. Structural studies demonstrate that this low affinity results from the obstruction of the enzyme active site by the side chain of Trp222, which presents a transient steric barrier to an incoming carbapenem substrate. The Trp222Met substitution relieves this steric hindrance and elevates the affinity of the mutant enzyme for carbapenems by 10-fold, significantly increasing the levels of resistance to these antibiotics. The ability of OXA-51 to evolve into a robust carbapenemase as the result of a single amino acid substitution may, in the near future, elevate the ubiquitous enzymes of the OXA-51 family to the status of the most deleterious A. baumannii carbapenemases, with dire clinical consequences.

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES β-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES β-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to an increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem-GES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 β-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES β-lactamases. Our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.

Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6')-Ie-APH(2'')-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6')-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2'')-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2'')-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6')-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6')-Ie enzyme is joined to APH(2'')-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2'')-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6')-Ie-APH(2'')-Ia enzyme.

The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are enzymes of the utmost clinical importance due to their ability to produce resistance to carbapenems, the antibiotics of last resort for the treatment of various life-threatening infections. The vast majority of these enzymes have been identified in Acinetobacter spp., notably in Acinetobacter baumannii. The OXA-2 and OXA-10 enzymes predominantly occur in Pseudomonas aeruginosa and are currently classified as narrow-spectrum class D β-lactamases. Here we demonstrate that when OXA-2 and OXA-10 are expressed in Escherichia coli strain JM83, they produce a narrow-spectrum antibiotic resistance pattern. When the enzymes are expressed in A. baumannii ATCC 17978, however, they behave as extended-spectrum β-lactamases and confer resistance to carbapenem antibiotics. Kinetic studies of OXA-2 and OXA-10 with four carbapenems have demonstrated that their catalytic efficiencies with these antibiotics are in the same range as those of some recognized class D carbapenemases. These results are in disagreement with the classification of the OXA-2 and OXA-10 enzymes as narrow-spectrum β-lactamases, and they suggest that other class D enzymes that are currently regarded as noncarbapenemases may in fact be CHDLs.

SGM-1 is a novel class A β-lactamase from an environmental isolate of Sphingobium sp. containing all of the distinct amino acid motifs of class A β-lactamases. It shares 77 to 80% amino acid sequence identity with putative β-lactamases that are present on the chromosome of all Sphingobium species whose genomes were sequenced and annotated. Thus, SGM-type β-lactamases are native to this genus. Antibiotic susceptibility testing classifies SGM-1 as an extended-spectrum β-lactamase, conferring the highest level of resistance to penicillins. Although SGM-1 contains the conserved cysteine residues characteristic of class A carbapenemases, it does not confer resistance to the carbapenem antibiotics imipenem, meropenem, or doripenem but does increase the MIC of ertapenem 8-fold. SGM-1 hydrolyzes penicillins and the monobactam aztreonam with similar catalytic efficiencies, ranging from 10(5) to 10(6) M(-1) s(-1). The catalytic efficiencies of SGM-1 for cefoxitin and ceftazidime were the lowest (10(2) to 10(3) M(-1) s(-1)) among the cephalosporins tested, while the catalytic efficiencies against all other cephalosporins varied from about 10(5) to 10(6) M(-1) s(-1). SGM-1 exhibited measurable but not significant activity toward the carbapenems tested. SGM-1 also showed high affinity for clavulanic acid, tazobactam, and sulbactam (Ki < 1 μM); however, only clavulanic acid significantly reduced the MICs of β-lactams.

Aminoglycoside 2″-phosphotransferases are the major aminoglycoside-modifying enzymes in clinical isolates of enterococci and staphylococci. We describe a novel aminoglycoside 2″-phosphotransferase from the Gram-negative pathogen Campylobacter jejuni, which shares 78% amino acid sequence identity with the APH(2″)-Ia domain of the bifunctional aminoglycoside-modifying enzyme aminoglycoside (6') acetyltransferase-Ie/aminoglycoside 2″-phosphotransferase-Ia or AAC(6')-Ie/APH(2″)-Ia from Gram-positive cocci, which we called APH(2″)-If. This enzyme confers resistance to the 4,6-disubstituted aminoglycosides kanamycin, tobramycin, dibekacin, gentamicin, and sisomicin, but not to arbekacin, amikacin, isepamicin, or netilmicin, but not to any of the 4,5-disubstituted antibiotics tested. Steady-state kinetic studies demonstrated that GTP, and not ATP, is the preferred cosubstrate for APH(2″)-If. The enzyme phosphorylates the majority of 4,6-disubstituted aminoglycosides with high catalytic efficiencies (k(cat)/K(m) = 10(5) to 10(7) M(-1) s(-1)), while the catalytic efficiencies against the 4,6-disubstituted antibiotics amikacin and isepamicin are 1 to 2 orders of magnitude lower, due mainly to the low apparent affinities of these substrates for the enzyme. Both 4,5-disubstituted antibiotics and the atypical aminoglycoside neamine are not substrates of APH(2″)-If, but are inhibitors. The antibiotic susceptibility and substrate profiles of APH(2″)-If are very similar to those of the APH(2″)-Ia phosphotransferase domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme.

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Carbapenem antibiotics have become therapeutics of last resort for the treatment of difficult infections. The emergence of class-A β-lactamases that have the ability to inactivate carbapenems in the past few years is a disconcerting clinical development in light of the diminished options for treatment of infections. A member of the GES-type β-lactamase family, GES-1, turns over imipenem poorly, but the GES-5 β-lactamase is an avid catalyst for turnover of this antibiotic. We report herein high-resolution X-ray structures of the apo GES-5 β-lactamase and the GES-1 and GES-5 β-lactamases in complex with imipenem. The latter are the first structures of native class-A carbapenemases with a clinically used carbapenem antibiotic in the active site. The structural information is supplemented by information from molecular dynamics simulations, which collectively for the first time discloses how the second step of catalysis by these enzymes, namely, hydrolytic deacylation of the acyl-enzyme species, takes place effectively in the case of the GES-5 β-lactamase and significantly less so in GES-1. This information illuminates one evolutionary path that nature has taken in the direction of the inexorable emergence of resistance to carbapenem antibiotics.

The class A carbapenemase KPC-6 produces resistance to a broad range of β-lactam antibiotics. This enzyme hydrolyzes penicillins, the monobactam aztreonam, and carbapenems with similar catalytic efficiencies, ranging from 10(5) to 10(6) M(-1) s(-1). The catalytic efficiencies of KPC-6 against cephems vary to a greater extent, ranging from 10(3) M(-1) s(-1) for the cephamycin cefoxitin and the extended-spectrum cephalosporin ceftazidime to 10(5) to 10(6) M(-1) s(-1) for the narrow-spectrum and some of the extended-spectrum cephalosporins.

FPH-1 is a new class A carbapenemase from Francisella philomiragia. It produces high-level resistance to penicillins and the narrow-spectrum cephalosporin cephalothin and hydrolyzes these β-lactam antibiotics with catalytic efficiencies of 10(6) to 10(7) M(-1) s(-1). When expressed in Escherichia coli, the enzyme confers resistance to clavulanic acid, tazobactam, and sulbactam and has K(i) values of 7.5, 4, and 220 μM, respectively, against these inhibitors. FPH-1 increases the MIC of the monobactam aztreonam 256-fold and the MIC of the broad-spectrum cephalosporin ceftazidime 128-fold, while the MIC of cefoxitin remains unchanged. MICs of the carbapenem antibiotics imipenem, meropenem, doripenem, and ertapenem are elevated 8-, 8-, 16-, and 64-fold, respectively, against an E. coli JM83 strain producing the FPH-1 carbapenemase. The catalytic efficiencies of the enzyme against carbapenems are in the range of 10(4) to 10(5) M(-1) s(-1). FPH-1 is 77% identical to the FTU-1 β-lactamase from Francisella tularensis and has low amino acid sequence identity with other class A β-lactamases. Together with FTU-1, FPH-1 constitutes a new branch of the prolific and ever-expanding class A β-lactamase tree.

Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen-deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232-252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of λ N (K(λN)) needed to achieve 50% maximal ATPase stimulation. Comparing the K(λN) values reveals that the region encompassing 232-252 of ELon could contribute to λ N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240-252 of ELon is important for ATPase activity, substrate translocation and protein degradation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2''-phosphotransferase IIIa (APH(2'')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2'')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.

The class A β-lactamase FTU-1 produces resistance to penicillins and ceftazidime but not to any other β-lactam antibiotics tested. FTU-1 hydrolyzes penicillin antibiotics with catalytic efficiencies of 10(5) to 10(6) M(-1) s(-1) and cephalosporins and carbapenems with catalytic efficiencies of 10(2) to 10(3) M(-1) s(-1), but the monobactam aztreonam and the cephamycin cefoxitin are not substrates for the enzyme. FTU-1 shares 21 to 34% amino acid sequence identity with other class A β-lactamases and harbors two cysteine residues conserved in all class A carbapenemases. FTU-1 is the first weak class A carbapenemase that is native to Francisella tularensis.

Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.

The Glu166Arg/Met182Thr mutant of Escherichia coli TEM(pTZ19-3) β-lactamase produces a 128-fold increase in the level of resistance to the antibiotic ceftazidime in comparison to that of the parental wild-type enzyme. The single Glu166Arg mutation resulted in a dramatic decrease in both the level of enzyme expression in bacteria and the resistance to penicillins, with a concomitant 4-fold increase in the resistance to ceftazidime, a third-generation cephalosporin. Introduction of the second amino acid substitution, Met182Thr, restored enzyme expression to a level comparable to that of the wild-type enzyme and resulted in an additional 32-fold increase in the minimal inhibitory concentration of ceftazidime to 64 μg/mL. The double mutant formed a stable covalent complex with ceftazidime that remained intact for the entire duration of the monitoring, which exceeded a time period of 40 bacterial generations. Compared to those of the wild-type enzyme, the affinity of the TEM(pTZ19-3) Glu166Arg/Met182Thr mutant for ceftazidime increased by at least 110-fold and the acylation rate constant was augmented by at least 16-fold. The collective experimental data and computer modeling indicate that the deacylation-deficient Glu166Arg/Met182Thr mutant of TEM(pTZ19-3) produces resistance to the third-generation cephalosporin ceftazidime by an uncommon covalent-trapping mechanism. This is the first documentation of such a mechanism by a class A β-lactamase in a manifestation of resistance., (© 2011 American Chemical Society)

The GES-2 β-lactamase is a class A carbapenemase, the emergence of which in clinically important bacterial pathogens is a disconcerting development as the enzyme confers resistance to carbapenem antibiotics. Tazobactam is a clinically used inhibitor of class A β-lactamases, which inhibits the GES-2 enzyme effectively, restoring susceptibility to β-lactam antibiotics. We have investigated the details of the mechanism of inhibition of the GES-2 enzyme by tazobactam. By the use of UV spectrometry, mass spectroscopy, and x-ray crystallography, we have documented and identified the involvement of a total of seven distinct GES-2·tazobactam complexes and one product of the hydrolysis of tazobactam that contribute to the inhibition profile. The x-ray structures for the GES-2 enzyme are for both the native (1.45 Å) and the inhibited complex with tazobactam (1.65 Å). This is the first such structure of a carbapenemase in complex with a clinically important β-lactam inhibitor, shedding light on the structural implications for the inhibition process.

Bacterial resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is commonly the result of the production of β-lactamases. The emergence of β-lactamases capable of turning over carbapenem antibiotics is of great concern, since these are often considered the last resort antibiotics in the treatment of life-threatening infections. β-Lactamases of the GES family are extended-spectrum enzymes that include members that have acquired carbapenemase activity through a single amino acid substitution at position 170. We investigated inhibition of the GES-1, -2, and -5 β-lactamases by the clinically important β-lactamase inhibitor clavulanic acid. While GES-1 and -5 are susceptible to inhibition by clavulanic acid, GES-2 shows the greatest susceptibility. This is the only variant to possess the canonical asparagine at position 170. The enzyme with asparagine, as opposed to glycine (GES-1) or serine (GES-5), then leads to a higher affinity for clavulanic acid (K(i) = 5 μM), a higher rate constant for inhibition, and a lower partition ratio (r ≈ 20). Asparagine at position 170 also results in the formation of stable complexes, such as a cross-linked species and a hydrated aldehyde. In contrast, serine at position 170 leads to formation of a long-lived trans-enamine species. These studies provide new insight into the importance of the residue at position 170 in determining the susceptibility of GES enzymes to clavulanic acid.

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2''-IVa (APH(2'')-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2'')-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k(cat)/K(m)) of 1.5 x 10(3) to 1.2 x 10(6) (M(-1) s(-1)). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2'')-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.

Directed evolution by random PCR mutagenesis of the gene for the aminoglycoside 2''-IIa phosphotransferase generated R92H/D268N and N196D/D268N mutant enzymes, resulting in elevated levels of resistance to amikacin and isepamicin but not to other aminoglycoside antibiotics. Increases in the activities of the mutant phosphotransferases for isepamicin are the result of decreases in K(m) values, while improved catalytic efficiency for amikacin is the result of both a decrease in K(m) values and an increase in turnover of the antibiotic. Enzymes with R92H, D268N, and D268N single amino acid substitutions did not result in elevated MICs for aminoglycosides.

We describe herein a highly proficient class A beta-lactamase OIH-1 from the bacterium Oceanobacillus iheyensis, whose habitat is the sediment at a depth of 1050 m in the Pacific Ocean. The OIH-1 structure was solved by molecular replacement and refined at 1.25 A resolution. OIH-1 has evolved to be an extremely halotolerant beta-lactamase capable of hydrolyzing its substrates in the presence of NaCl at saturating concentration. Not only is this the most highly halotolerant bacterial enzyme structure known to date, it is also the highest resolution halophilic protein structure yet determined. Evolution of OIH-1 in the salinity of the ocean has resulted in a molecular surface that is coated with acidic residues, a marked difference from beta-lactamases of terrestrial sources. OIH-1 is the first example of an antibiotic-resistance enzyme that has evolved in the depths of the ocean in isolation from clinical selection and gives us an extraordinary glimpse into protein evolution under extreme conditions. It represents evidence for the existence of a reservoir of antibiotic-resistance enzymes in nature among microbial populations from deep oceanic sources.

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.

Lon, also known as protease La, is an ATP-dependent protease functioning to degrade many unstructured proteins. Currently, very little is known about the substrate determinants of Lon at the proteolytic site. Using synthetic peptides constituting different regions of the endogenous protein substrate lambdaN, we demonstrated that the proteolytic site of Escherichia coli Lon exhibits a certain level of localized sequence specificity. Using an alanine positional scanning approach, we discovered a set of discontinuous substrate determinants surrounding the scissile Lon cleavage site in a model peptide substrate, which function to influence the k(cat) of the peptidase activity of Lon. We further investigated the mode of peptide interaction with the proteolytically inactive Lon mutant S679A in the absence and presence of ADP or AMPPNP by 2-dimensional nuclear magnetic resonance spectroscopy, and discovered that the binding interaction between protein and peptide varies with the nucleotide bound to the enzyme. This observation is suggestive of a substrate translocation step, which likely limits the turnover of the proteolytic reaction. The contribution of the identified substrate determinants towards the kinetics of ATP-dependent degradation of lambdaN and truncated lambdaN mutants by Lon was also examined. Our results indicated that Lon likely recognizes numerous discontinuous substrate determinants throughout lambdaN to achieve substrate promiscuity.

Lon is a homo-oligomeric ATP-dependent serine protease that functions in the degradation of damaged and certain regulatory proteins. This enzyme has emerged as a novel target in the development of antibiotics because of its importance in conferring bacterial virulence. In this study, we explored the mechanism by which the proteasome inhibitor MG262, a peptidyl boronate, inhibits the peptide hydrolysis activity of Salmonella enterica serovar Typhimurium Lon. In addition, we synthesized a fluorescent peptidyl boronate inhibitor based upon the amino acid sequence of a product of peptide hydrolysis by the enzyme. Using steady-state kinetic techniques, we have shown that two peptidyl boronate variants are competitive inhibitors of the peptide hydrolysis activity of Lon and follow the same two-step, time-dependent inhibition mechanism. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. This is followed by a second slow step in which Lon undergoes a conformational change or isomerization to increase the interaction of the inhibitor with the proteolytic active site to yield an overall inhibition constant of 5-20 nM. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease that has required the binding of ATP in order to observe inhibition.