Structural insights into the broadened substrate profile of the extended-spectrum β-lactamase OXY-1-1 fromKlebsiella oxytoca

Klebsiella oxytocais a pathogen that causes serious infections in hospital patients. It shows resistance to many clinically used β-lactam antibiotics by producing chromosomally encoded OXY-family β-lactamases. Here, the crystal structure of an OXY-family β-lactamase, OXY-1-1, determined at 1.93 Å resolution is reported. The structure shows that the OXY-1-1 β-lactamase has a typical class A β-lactamase fold and exhibits greater similarity to CTX-M-type β-lactamases than to TEM-family or SHV-family β-lactamases. It is also shown that the enzyme provides more space around the active cavity for theR1andR2substituents of β-lactam antibiotics. The half-positive/half-negative distribution of surface electrostatic potential in the substrate-binding pocket indicates the preferred properties of substrates or inhibitors of the enzyme. The results reported here provide a structural basis for the broadened substrate profile of the OXY-family β-lactamases.

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Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0905270106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15616-15621 ◽

Cited By ~ 30

Author(s):

Masataka Umitsu ◽

Hiroshi Nishimasu ◽

Akiko Noma ◽

Tsutomu Suzuki ◽

Ryuichiro Ishitani ◽

...

Keyword(s):

Crystal Structures ◽

Binding Pocket ◽

Structural Basis ◽

Binding Modes ◽

Substrate Binding Pocket ◽

Methyl Group ◽

Group Transfer ◽

Canonical Class ◽

Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

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The Structural Basis for Glycerol Permeation by human AQP7

10.1101/858332 ◽

2019 ◽

Cited By ~ 1

Author(s):

Li Zhang ◽

Deqiang Yao ◽

Fu Zhou ◽

Qing Zhang ◽

Ying Xia ◽

...

Keyword(s):

Physiological Condition ◽

Critical Role ◽

Binding Pocket ◽

Structural Basis ◽

Glycerol Metabolism ◽

Functional Assay ◽

Substrate Binding Pocket ◽

Glycerol Molecule ◽

Cytoplasmic Side

AbstractHuman glycerol channel AQP7 conducts glycerol release from adipocyte and entry into the cells in pancreatic islets, muscles and kidney tubule, and thus regulate glycerol metabolism in those tissues. Compared with other human aquaglyceroporins, AQP7 shows a less conserved “NPA” motif in the center cavity, and a pair of aromatic residues at Ar/R selectivity filter. To understand the structural basis for the glycerol conductance, we crystallized the human AQP7 and determined the structure at 3.7 Å. A substrate binding pocket was found near to the Ar/R filter and the bound glycerol molecule stabilized by R229. In vivo functional assay on human AQP7 as well as AQP3 and AQP10 demonstrated strong glycerol transportation activities at physiological condition. The human AQP7 structure reveals a fully closed conformation with its permeation pathway strictly confined by Ar/R filter at the exoplasmic side and the gate at the cytoplasmic side, and the dislocation of the residues at narrowest parts of glycerol pathway in AQP7 play a critical role in controlling the glycerol flux.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113573118 ◽

2021 ◽

Vol 118 (49) ◽

pp. e2113573118

Author(s):

Carlos F. Rodriguez ◽

Paloma Escudero-Bravo ◽

Lucía Díaz ◽

Paola Bartoccioni ◽

Carmen García-Martín ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Substrate Specificity ◽

Molecular Mechanisms ◽

Substrate Binding ◽

Binding Pocket ◽

Neutral Amino Acid ◽

Substrate Preference ◽

Structural Basis ◽

Substrate Binding Pocket

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.

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

Biochemical Journal ◽

10.1042/bcj20210647 ◽

2021 ◽

Author(s):

X Edward Zhou ◽

Kelly Suino-Powell ◽

Chad R Schultz ◽

Bilal Aleiwi ◽

Joseph S Brunzelle ◽

...

Keyword(s):

Crystal Structure ◽

Mass Spectrometry ◽

Ornithine Decarboxylase ◽

Complex Structure ◽

Binding Pocket ◽

Catalytic Site ◽

Structural Basis ◽

High Potency ◽

Substrate Binding Pocket ◽

Rate Limiting

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.

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Structural basis for active-site probes targeting Staphylococcus aureus serine hydrolase virulence factors

10.1101/2020.04.21.054437 ◽

2020 ◽

Author(s):

Matthias Fellner ◽

Christian S. Lentz ◽

Sam A. Jamieson ◽

Jodi L. Brewster ◽

Linhai Chen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Active Site ◽

Binding Pocket ◽

Structural Basis ◽

Serine Hydrolase ◽

Serine Hydrolases ◽

Substrate Binding Pocket ◽

Hydrophobic Substrate ◽

Substrate Analog ◽

Insight Into

SummaryStaphylococcus aureus is a major cause of infection in the community and in hospitals. Serine hydrolases play key roles in bacterial homeostasis, in particular biofilms. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fphs), which contribute to virulence of S. aureus in the biofilm niche. Here we report structures of the putative tributyrin esterase FphF, alone and covalently bound by a substrate analog, and small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. We show that FphF has promiscuous esterase activity. Building from this, we extended our analysis to the wider Fph protein family using homology modeling and docking tools. We predict that other Fph enzymes, including FphB which was linked directly to virulence, may be more specific than FphF. This study provides insight into Fph function and a template for designing new imaging agents, diagnostic probes, and inhibitors to treat S. aureus infections.

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Structural Insights into Azole-based Inhibitors of Heme Oxygenase-1: Development of Selective Compounds for Therapeutic Applications

Current Medicinal Chemistry ◽

10.2174/0929867325666180606082512 ◽

2019 ◽

Vol 25 (42) ◽

pp. 5803-5821 ◽

Cited By ~ 3

Author(s):

Mona N. Rahman ◽

Dragic Vukomanovic ◽

Jason Z. Vlahakis ◽

Walter A. Szarek ◽

Kanji Nakatsu ◽

...

Keyword(s):

Heme Oxygenase ◽

Competitive Inhibition ◽

Cytochromes P450 ◽

Structural Similarity ◽

Binding Pocket ◽

Initial Study ◽

Structural Basis ◽

Heme Oxygenase 1 ◽

Design Strategies ◽

Inhibitor Binding

The development of isozyme-selective heme oxygenase (HO) inhibitors promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties with a role in several disease states; thus, it is an enticing therapeutic target. Historically, the metalloporphyrins have been used as competitive HO inhibitors based on their structural similarity to the substrate, heme. However, heme’s important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), results in non-selectivity being an unfortunate side effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort over a decade ago to develop novel compounds as potent, selective inhibitors of HO. The result was the creation of the first generation of non-porphyrin based, non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated and provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. Notably, HO-1 inhibitors are of particular interest for the treatment of hyperbilirubinemia and certain types of cancer. Key features based on this initial study have already been used by others to discover additional potential HO-1 inhibitors. Moreover, studies have begun to use selected compounds and determine their effects in some disease models.

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Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

Nature Communications ◽

10.1038/s41467-020-20675-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yufei Han ◽

Qian Zhuang ◽

Bo Sun ◽

Wenping Lv ◽

Sheng Wang ◽

...

Keyword(s):

Crystal Structure ◽

Biochemical Characterization ◽

Substrate Binding ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Transmembrane Segments ◽

Therapeutic Molecules ◽

Binding Cavity ◽

Immune System Regulation ◽

Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

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