Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

Halo-Tolerant And Thermostable Mechanisms of An Endoglucanase From Marine Aspergillus Niger

The cellulase cocktail of marine Aspergillus niger exhibited halo-tolerant and thermostable properties, which is of great potential in industrial application. In order to excavate the single tolerant cellulase components from complex cellulase cocktail, constitutive homologous expression was employed for direct obtainment of the endoglucanase (AnEGL). Enzymatic property study revealed that AnEGL exhibited a property of halo-tolerance and an outstanding thermostability in high salinity environment. Significantly, its activity increased by 29% and the half-life at 65 °C increased by 26.7-fold with the presence of 4.5 M NaCl. Molecular dynamics simulation revealed that Na+ and Cl－ could form salt bridges with charged residues, and then influenced the activity of loops and the stability of substrate binding pocket, which accounted for the halo-tolerance and thermostability. Further, site-specific mutagenesis study proved that the residues Asp95 and Asp99 in the pocket were of great concern for the tolerant properties. The halo-tolerant and thermostable AnEGL was of great value in lignocellulosic utilization and the conjectural mechanisms were of referential significance for other tolerant enzymes.

Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC–APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Å resolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA’s high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.

Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.

Transport Efficiency of AtGTR1 Dependents on the Hydrophobicity of Transported Glucosinolates

Glucosinolates (GLSs) are a group of secondary metabolites that are involved in the defense of herbivores. In Arabidopsis thaliana, Glucosinolate Transporter 1 (AtGTR1) transports GLSs with high affinity via a proton gradient-driven process. In addition to transporting GLSs, AtGTR1 also transports phytohormones, jasmonic acidisoleucine (JA-Ile), and gibberellin (GA). However, little is known about the mechanisms underlying the broad substrate specificity of AtGTR1. Here, we characterized the substrate preference of AtGTR1 by using a yeast uptake assay, and the results revealed that GLS transport rates are negatively correlated with the hydrophobicity of substrates. Interestingly, the AtGTR1 showed a higher substrate affinity for GLSs with higher hydrophobicity, suggesting a hydrophobic substrate binding pocket. In addition, a competition assay revealed that the presence of JA, salicylic acid (SA), and indole-3-acetic acid (IAA) inhibits the transport of GLSs in yeast, suggesting a potential regulatory mechanism of AtGTR1. To further characterize the functional properties of AtGTR1, mutagenesis experiments confirmed that the conserved EXXEK motif and Arg166 are essential for the GLS transport function. In addition, the purified AtGTR1 adopts a homodimeric conformation, which is possibly regulated by phosphorylation on Thr105. The phosphomimetic mutation, T105D, reduced its protein expression and completely abrogated its GLS transport function, indicating the essential role of phosphorylation on AtGTR1. In summary, this study investigated various factors associated with the GLS transport and increased our knowledge on the substrate preferences of AtGTR1. These findings contribute to understanding how the distribution of defense GLSs is regulated in plants and could be used to improve crop quality in agriculture.

Deep mutational scan of a drug efflux pump reveals its structure-function landscape

Drug efflux is a common resistance mechanism found in bacteria and cancer cells. Although several structures of drug efflux pumps are available, they provide only limited functional information on the phenomenon of drug efflux. Here, we performed deep mutational scanning (DMS) on the bacterial ATP binding cassette (ABC) transporter EfrCD to determine the drug efflux activity profile of more than 1500 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity towards positively charged ethidium, while additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryo-EM structure of EfrCD uncovered a high affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert the ABC exporter EfrCD into a drug-specific ABC importer.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.