A comparative analysis of β-mannanases of bacteria from Antarctica and Malaysia

β-mannanase is an enzyme that is commonly expressed in environmental bacteria. It degrades hemicellulose found in plant material and recycles nutrients back into the environment. Because this enzyme significantly contributes to biodegradation and has recently been applied in industry, we conducted a comparative analysis of bacterial isolates found in soil samples from Schirmacher Oasis, Antarctica, and Sabah, Malaysia that were capable of degrading mannan. A total of 9 bacterial isolates from Antarctica and 30 bacterial isolates from Malaysia exhibited β-mannanase activity. These bacteria were differentiated and clustered using their random amplified polymorphic DNA (RAPD) profiles, and the β-mannanase activity of these isolates was tested at different temperatures and pH. Five out of 9 Antarctica isolates and seven out of 30 Malaysian isolates were identified based on their 16S rDNA sequences. Identified bacterial isolates from Antarctica were: MP1 (Bacillus amyloliquefaciens), MP2 (Bacillus pumilus), MP5 (Bacillus pumilus), A40 (Arthrobacter sp.), and C27 (Arthrobacter oxydans). Identified bacterial isolates from Malaysia were: Y1 (Paenibacillus sp.), Y2 (Bacillus sp.), Y16 (Paenibacillus sp.), Y18 (Paenibacillus sp.), A7 (Paenibacillus sp.), B26 (Streptomyces sp.), and D4 (Paenibacillus amylolyticus). β-mannanases produced by the Antarctica bacterial isolates MP1 (Bacillus amyloliquefaciens) and A40 (Arthrobacter sp.) were active at 5℃ and 20℃, respectively, while the β-mannanase produced by the bacterial isolate from Malaysia, A7 (Paenibacillus sp.), was active at 35℃

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Characterization of antarctic bacteria and their antimicrobial activities

A total of 2582 bacterial strains were isolated from 16 soil and water samples from the King George Island and Schirmacher Range, Antarctica. Twenty three Antarctic bacterial strains inhibited the growth of one or more Gram-negative and Grampositive food pathogens such as Escherichia coli O 157: H7, Salmonella spp., Klebsiella pneumonia, Enterobacter cloacae and Vibrio spp. and a Gram-positive food pathogen Bacillus cereus K15. Seven out of the 23 strains, SGS, CG21, HKAM1, MTC3, MA2, WEAl and WEKl were identified based on their 16S rDNA sequences and biochemical analyses. They were Pseudomonas sp. MTC3, Pseudomonas sp. CG21, Pseudomonas sp. MA2, P. corrugata WEKl, P. migulae WEAl, Janthinobacterium lividum HKAMl and Pedobacter cryoconits SGS. Although most of them were affiliated to the same genus or closely related species, their biochemical, phenotypic characteristics and antibiotics resistance profiles varied. Inhibitors produced by strains MTC3, CG21 and SGS were sensitive to protease suggesting that they have proteinaceous structures while strains WEAl, WEKl, HKAMl and MA2 were not sensitive to catalase, lipase, aamylase, and protease indicating four of these inhibitors have complex structures. Three out of seven Antarctic bacterial strains WEAl, WEK1 and MA2 were found to encode polyketide synthase gene, indicating the antimicrobial agent was probably produced by polyketide synthase. Antimicrobial resistance profiles of 45 Antarctic bacterial isolates were obtained. Most of the bacteria were resistance to at least of three or more types of antimicrobial agents tested while one of the bacterial isolate was susceptible to all the antimicrobial agents. These data revealed that the existence of many antimicrobial resistant strains among the Antarctic bacterial population. The plasmid sequence of pH Kl of Pseudomonas sp. CG21 revealed that there was no gene encoding the antimicrobial production and antimicrobial resistance on the plasmid. Basically the pHKl plasmid carried genes encoding for plasmid replication, stability and maintenance, mobilization and genes for unknown function

Binding of tetracyclines to Acinetobacter baumannii TetR involves two arginines as specificity determinants

Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii

Allosteric drug transport mechanism of multidrug transporter AcrB

Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs

Exploring the Molecular Linkage of Protein Stability Traits for Enzyme Optimization by Iterative Truncation and Evolution

The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation–compensation strategy, which identifies mutations rescuing the activity of a truncated TEM β-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C in <i>T</i>_m, displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a <i>T</i>_m increased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation–optimization cycles can exploit stability–trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information

A new critical conformational determinant of multidrug efflux by an MFS transporter

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state

A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state