Tebipenem, a New Carbapenem Antibiotic, Is a Slow Substrate That Inhibits the β‑Lactamase from <i>Mycobacterium tuberculosis</i>

The genome of Mycobacterium tuberculosis contains a gene, <i>blaC</i>, which encodes a highly active β-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme–drug covalent complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent <i>K</i>_m and <i>k</i>_cat values of 0.8 μM and 0.03 min^–1, respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tight-binding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl–enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC–tebipenem covalent acylated adduct (1.9 Å) with wild-type BlaC and the BlaC–tebipenem Michaelis–Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem–BlaC structures

Understanding the basis of thermostability for enzyme “Nanoluc” towards designing industry-competent engineered variants

As a leading contender in the study of luminescence, nanoluciferase has recently attracted attention and proven effective in a wide variety of research areas. Although numerous attempts have been made to improve activity, there has yet to be a thorough exploration of further possibilities to improve thermostability. In this study, protein engineering in tandem with molecular dynamics simulation at various temperatures (300 K, 400 K, 450 K and 500 K) was used to improve our understanding of nanoluciferase dynamics and identification of factors that could significantly enhance the thermostability. Based on these, three novel mutations have been narrowed down, which were hypothesised to improve thermostability. Root mean square deviation and root mean square fluctuation studies confirmed higher stability of mutant at high temperature. Solvent-accessible surface area and protein unfolding studies revealed a decreased tendency of mutant to unfold at higher temperatures. Further free energy landscape and principal component analysis was adapted to get deeper insights into the thermodynamic and structural behavior of these proteins at elevated temperature. Thus, this study provides a deeper insight into the dynamic factors for thermostability and introduces a novel, enhanced nanoluciferase candidate with potential use in industry. Communicated by Ramaswamy H. Sarma</p

NXL104 Irreversibly Inhibits the β-Lactamase from <i>Mycobacterium tuberculosis</i>

NXL104 is a novel β-lactamase inhibitor with a non-lactam structural scaffold. Our kinetic and mass spectrometric analysis demonstrates that NXL104 quantitatively inhibits BlaC, the only chromosomally encoded β-lactamase from <i>Mycobacterium tuberculosis</i>, by forming a carbamyl adduct with the enzyme. The inhibition efficiency (<i>k</i>₂/<i>K</i>) of NXL104 was shown to be more than 100-fold lower than that of clavulanate, a classical β-lactamase inhibitor, which is probably caused by the bulky rings of NXL104. However, the decarbamylation rate constant (<i>k</i>₃) was determined to be close to zero. The BlaC–NXL104 adduct remained stable for at least 48 h, while the hydrolysis of the BlaC–clavulanate adduct was observed after 2 days. The three-dimensional crystal structure of the BlaC–-NXL104 carbamyl adduct was determined at a resolution of 2.3 Å. Interestingly, the sulfate group of NXL104 occupies the position of a phosphate ion in the structure of the BlaC–clavulanate adduct and is hydrogen bonded to residues Ser128, Thr237, and Thr239. Favorable interactions are also seen in the electrostatic potential map. We propose that these additional interactions, as well as the intrinsic stability of the carbamyl linkage, contribute to the extraordinary stability of the BlaC–NXL104 adduct

Structural, Kinetic and Chemical Mechanism of Isocitrate Dehydrogenase‑1 from <i>Mycobacterium tuberculosis</i>

<i>Mycobacterium tuberculosis</i> (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In <i>Escherichia coli</i>, flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently, there is no mechanistic or structural information about ICDH. We optimized expression and purification to a yield sufficiently high to perform the first detailed kinetic and structural studies of Mtb ICDH-1. A large solvent kinetic isotope effect [^D₂O<i>V</i> = 3.0 ± 0.2, and ^D₂O(<i>V</i>/<i>K</i>_isocitrate) = 1.5 ± 0.3] and a smaller primary kinetic isotope effect [^D<i>V</i> = 1.3 ± 0.1, and ^D(<i>V</i>/<i>K</i>_{[2<i>R</i>‑²H]isocitrate}) = 1.5 ± 0.2] allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn²⁺ bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking β sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyzes the formation of α-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel

Structure of MurNAc 6‑Phosphate Hydrolase (MurQ) from <i>Haemophilus influenzae</i> with a Bound Inhibitor

The breakdown and recycling of peptidoglycan, an essential polymeric cell structure, occur in a number of bacterial species. A key enzyme in the recycling pathway of one of the components of the peptidoglycan layer, <i>N</i>-acetylmuramic acid (MurNAc), is MurNAc 6-phosphate hydrolase (MurQ). This enzyme catalyzes the cofactor-independent cleavage of a relatively nonlabile ether bond and presents an interesting target for mechanistic studies. Open chain product and substrate analogues were synthesized and tested as competitive inhibitors (<i>K</i>_is values of 1.1 ± 0.3 and 0.23 ± 0.02 mM, respectively) of the MurNAc 6P hydrolase from <i>Escherichia coli</i> (MurQ-EC). To identify the roles of active site residues that are important for catalysis, the substrate analogue was cocrystallized with the MurNAc 6P hydrolase from <i>Haemophilus influenzae</i> (MurQ-HI) that was amenable to crystallographic studies. The cocrystal structure of MurQ-HI with the substrate analogue showed that Glu89 was located in the proximity of both the C2 atom and the oxygen at the C3 position of the bound inhibitor and that no other potential acid/base residue that could act as an active site acid/base was located in the vicinity. The conserved residues Glu120 and Lys239 were found within hydrogen bonding distance of the C5 hydroxyl group and C6 phosphate group, suggesting that they play a role in substrate binding and ring opening. Combining these results with previous biochemical data, we propose a one-base mechanism of action in which Glu89 functions to both deprotonate at the C2 position and assist in the departure of the lactyl ether at the C3 position. This same residue would serve to deprotonate the incoming water and reprotonate the enolate in the second half of the catalytic cycle

In Silico Designing of an Industrially Sustainable Carbonic Anhydrase Using Molecular Dynamics Simulation

Carbonic anhydrase (CA) is a family of metalloenzymes that has the potential to sequestrate carbon dioxide (CO₂) from the environment and reduce pollution. The goal of this study is to apply protein engineering to develop a modified CA enzyme that has both higher stability and activity and hence could be used for industrial purposes. In the current study, we have developed an in silico method to understand the molecular basis behind the stability of CA. We have performed comparative molecular dynamics simulation of two homologous α-CA, one of thermophilic origin (<i>Sulfurihydrogenibium</i> sp.) and its mesophilic counterpart (Neisseria gonorrhoeae), for 100 ns each at 300, 350, 400, and 500 K. Comparing the trajectories of two proteins using different stability-determining factors, we have designed a highly thermostable version of mesophilic α-CA by introducing three mutations (S44R, S139E, and K168R). The designed mutant α-CA maintains conformational stability at high temperatures. This study shows the potential to develop industrially stable variants of enzymes while maintaining high activity

Single Cell Oil from Oleaginous Yeast Grown on Sugarcane Bagasse-Derived Xylose: An Approach toward Novel Biolubricant for Low Friction and Wear

Yeast lipid as single cell oil (SCO) is evaluated as an alternative renewable source of vegetable oils for a biolubricant formulation. The <i>Rhodotorula mucilaginosa</i> IIPL32 yeast strain is cultivated on lignocellulosic pentosans derived from sugarcane bagasse to produce the SCO. The chemical composition and distribution of variable fatty acids in the yeast SCO are characterized by NMR, FTIR, and GC × GC analyses. The high viscosity index and a low pour point of yeast SCO owing to the favorable composition of saturated and unsaturated fatty acids promise its potential as a renewable and environmentally friendly lube base oil. The yeast SCO as lube base oil significantly reduced the coefficient of friction (72%) and wear (24%) compared to those of conventional mineral lube base oil (SN 150). The fatty acids in the yeast SCO formed a good quality tribo-chemical thin film on the engineering surfaces, which not only reduced the friction but also protected the contact interfaces against wear. This study demonstrates that yeast SCO being renewable, biodegradable, and nontoxic, provides favorable physicochemical and tribophysical properties for good quality lubricant formulation and it can be a good alternative to the conventional mineral lube oil-based lubricants