Abstractβ-lactam antibiotics are ineffective against Mycobacterium tuberculosis, being rapidly hydrolyzed by the chromosomally encoded blaC gene product. The carbapenem class of β-lactams are very poor substrates for BlaC, allowing us to determine the three-dimensional structure of the covalent BlaCmeropenem covalent complex at 1.8 angstrom resolution. When meropenem was combined with the β-lactamase inhibitor clavulanate, potent activity against laboratory strains of M. tuberculosis was observed [minimum inhibitory concentration (MIC meropenem ) less than 1 microgram per milliliter], and sterilization of aerobically grown cultures was observed within 14 days. In addition, this combination exhibited inhibitory activity against anaerobically grown cultures that mimic the "persistent" state and inhibited the growth of 13 extensively drug-resistant strains of M. tuberculosis at the same levels seen for drug-susceptible strains. Meropenem and clavulanate are Food and Drug Administration-approved drugs and could potentially be used to treat patients with currently untreatable disease.

Despite the enormous success of β-lactams as broad-spectrum antibacterials, they have never been widely used for the treatment of TB due to intrinsic resistance that is caused by the presence of a chromosomally-encoded gene (blaC) in Mycobacterium tuberculosis. Our previous studies of TB BlaC revealed that this enzyme is an extremely broad-spectrum β-lactamase hydrolyzing all β-lactam classes. Carbapenems are slow substrates that acylate the enzyme but are only slowly deacylated and can therefore act also as potent inhibitors of BlaC. We carried out the in vitro characterization of doripenem and ertapenem with BlaC. A steady-state kinetic burst was observed with both compounds with magnitudes proportional to the concentration of BlaC used. The results show apparent K m and k cat values of 0.18 µM and 0.016 min −1 for doripenem and 0.18 µM and 0.017 min −1 for ertapenem. FTICR mass spectrometry demonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The BlaCdoripenem covalent complex obtained after 90 minutes of soaking was solved to 2.2 Å, while the BlaC-ertapenem complex obtained after a 90 minute soak was solved to 2.0 Å. The 1.3 Å diffraction data from a 10 minute ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original Δ 2 pyrroline ring was tautomerized to generate the Δ 1 pyrroline ring. The isomerization leads to the flipping of the carbapenem-hydroxyethyl group to hydrogen bond to the carboxyl O2 of Glu166. The hydroxyethyl flip results in both decreased basicity of Glu166 and in a significant increase in the distance between the carboxyl O2 of Glu166 and the catalytic water molecule, slowing hydrolysis. Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a worldwide health concern (1). There were an estimated 9.3 million new cases of TB in 2007 and approximately 1.3 million HIV-negative patient fatalities as well as nearly half a million deaths amongst HIV-positive populations (2). Even fifty years after the introduction of powerful antibiotics to treat TB, it has been estimated that one person is infected in the world every few seconds (3). The failure to control TB is due to the emergence of M. tuberculosis strains that are multiply drug resistant towards the front line antimycobacterial drugs such as isoniazid and rifampicin.Phone: (718) Fax: (718) As one of the most important antibiotic families, β-lactams include a broad range of molecules including penicillin derivatives, cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors. The carbapenems exhibit the broadest spectrum of activity among the β-lactam antimicrobials, providing safe and efficacious therapies in the treatment of serious infections caused by Gram-positive, Gram-negative, and anaerobic bacterial pathogens (4,5). Carbapenem antibiotics were originally developed from thienamycin, a natural product identified in culture filtrates of Streptomyces cattleya (6). There are four carbapen...

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

BackgroundEver since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases.ResultsHere, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s.ConclusionsMISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0524-5) contains supplementary material, which is available to authorized users.

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 A with R(work) = 19.8% and R(free) = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with k(cat)/K(m) = 3.12 x 10(4) and 1.28 x 10(4) microM(-)(1) s(-)(1) for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k(cat)/K(m)) are low (1 x 10(4) M(-)(1) s(-)(1)) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.

The intrinsic resistance of Mycobacterium tuberculosis to the beta-lactam class of antibiotics arises from a chromosomally encoded, extended spectrum, class A beta-lactamase, BlaC. Herein, we report the X-ray crystallographic structure of BlaC inhibited with clavulanate at a resolution of 1.7 A with an R-factor value of 0.180 and R-free value of 0.212 for the m/ z +154 clavulanate-derived fragment observed in the active site. Structural evidence reveals the presence of hydrogen bonds to the C1 carbonyl along with a coplanar arrangement of C1, C2, C3, and N4, which favors enolization to generate a trans-alpha,beta-eneamine, stabilizing the +154 adduct from hydrolysis. The irreversible inhibition of BlaC suggests that treatment of M. tuberculosis with a combination of a beta-lactam antibiotic and clavulanate may lead to rapid bactericidal activity.

The current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal ␤-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosis strains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosis BlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitro clavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosis H37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to ␤-lactam-␤-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.