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

2022 ◽  
Vol 12 ◽  
Author(s):  
Yeongjin Yun ◽  
Sangjun Han ◽  
Yoon Sik Park ◽  
Hyunjae Park ◽  
Dogyeong Kim ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Chemical Structure ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Zinc Ions ◽  
Substrate Binding Pocket ◽  
Inhibitory Spectrum

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.

Expanding Substrate Specificity of ω-Transaminase by Rational Remodeling of a Large Substrate-Binding Pocket

2015 ◽  
Vol 357 (12) ◽  
pp. 2712-2720 ◽  
Author(s):  
Sang-Woo Han ◽  
Eul-Soo Park ◽  
Joo-Young Dong ◽  
Jong-Shik Shin
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket

scholarly journals Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

2005 ◽  
Vol 389 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Melinda DEMENDI ◽  
Noboru ISHIYAMA ◽  
Joseph S. LAM ◽  
Albert M. BERGHUIS ◽  
Carole CREUZENET
Keyword(s):  
Enzyme Activity ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Structural Data ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Expected Effect ◽  
Biochemical Assays ◽  
Golden Opportunity

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

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

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.

Targeting the Genetic Resistance of JAK2 and BCR/ABL by TG101348

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3765-3765
Author(s):  
Meenu Kesarwani ◽  
Mohammad Azam
Keyword(s):  
Active Site ◽  
Myeloproliferative Neoplasms ◽  
Substrate Binding ◽  
Genetic Resistance ◽  
Structural Modeling ◽  
Binding Pocket ◽  
Janus Kinase ◽  
Substrate Binding Pocket ◽  
Modeling Studies

Abstract Abstract 3765 The inherent preponderance of genetic resistance against tyrosine kinase inhibitor (TKI) therapy poses significant challenge for effective treatments. Recent approval of Janus kinase 2 (JAK2) inhibitor INCB018424 (Jakafi, ruxolitinib) for the treatment of myeloproliferative neoplasms (MPNs) prompted us to identify resistant mutations that may pose clinical challenge. In vitro drug selection screening against two leading JAK2 inhibitors (INCB018424 and TG101348) were performed. Here we show that like other kinase inhibitors, INCB018424 is prone to genetic resistance while TG101348 is recalcitrant to develop in vitro resistance. Sequencing of INCB018424 resistant clones identified 211 amino acid substitutions spanning across FERM, SH2, JH2 and the kinase domain. Biochemical and structural modeling studies of these mutants demonstrate that mutations within the active site confer resistance either by direct steric hindrance or destabilizing the architecture of the active site. And mutations from the allosteric sites destabilize the intermediary state of the active and inactive conformations of JAK2 to which INCB018424 preferentially binds. Furthermore, these resistant variants are cross resistant to other JAK2 inhbitors (Lestaurtinib, CYT-387 and AZD1480). In contrast, these resistant variants are fully sensitive to TG101348 supporting the lack of resistance against this compound as observed during in vitro screening. Structural modeling studies revealed that TG101348 stabilizes the active conformation of the kinase and binds to the substrate-binding pocket. Mutations affecting the substrate-binding pocket may either alter substrate binding/phosphorylation or encode an incompetent kinase that blocks the emergence of resistance. Because JAK2 and BCR/ABL share a common substrate, STAT5, they might have similar architecture of the substrate-binding pocket, which may allow the inhibition of BCR/ABL by TG101348. Indeed, TG101348 can inhibit both native and gatekeeper variants of BCR/ABL and in vitro drug resistant screening failed to develop emergence of resistant clones. These studies provide evidence that the patients developing resistant variants of JAK2 and BCR/ABL can be treated with TG101348 and support for future drug design geared towards targeting the substrate binding sites in other oncogenic kinases for better and sustained therapeutic response. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Catalytic Efficiency of Basidiomycete Laccases: Redox Potential versus Substrate-Binding Pocket Structure

Catalysts ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 152 ◽  
Author(s):  
Olga Glazunova ◽  
Nikita Trushkin ◽  
Konstantin Moiseenko ◽  
Ivan Filimonov ◽  
Tatyana Fedorova
Keyword(s):  
Redox Potential ◽  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Structural Features ◽  
Redox Potentials ◽  
Substrate Binding Pocket ◽  
Trametes Hirsuta ◽  
Special Cases ◽  
Almost All

Laccases are copper-containing oxidases that catalyze a one-electron abstraction from various phenolic and non-phenolic compounds with concomitant reduction of molecular oxygen to water. It is well-known that laccases from various sources have different substrate specificities, but it is not completely clear what exactly provides these differences. The purpose of this work was to study the features of the substrate specificity of four laccases from basidiomycete fungi Trametes hirsuta, Coriolopsis caperata, Antrodiella faginea, and Steccherinum murashkinskyi, which have different redox potentials of the T1 copper center and a different structure of substrate-binding pockets. Enzyme activity toward 20 monophenolic substances and 4 phenolic dyes was measured spectrophotometrically. The kinetic parameters of oxidation of four lignans and lignan-like substrates were determined by monitoring of the oxygen consumption. For the oxidation of the high redox potential (>700 mV) monophenolic substrates and almost all large substrates, such as phenolic dyes and lignans, the redox potential difference between the enzyme and the substrate (ΔE) played the defining role. For the low redox potential monophenolic substrates, ΔE did not directly influence the laccase activity. Also, in the special cases, the structure of the large substrates, such as dyes and lignans, as well as some structural features of the laccases (flexibility of the substrate-binding pocket loops and some amino acid residues in the key positions) affected the resulting catalytic efficiency.

scholarly journals Structural analysis of a function-associated loop mutant of the substrate-recognition domain of Fbs1 ubiquitin ligase

2016 ◽  
Vol 72 (8) ◽  
pp. 619-626 ◽  
Author(s):  
Kazuya Nishio ◽  
Yukiko Yoshida ◽  
Keiji Tanaka ◽  
Tsunehiro Mizushima
Keyword(s):  
Ubiquitin Ligase ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Degradation Pathway ◽  
Substrate Binding Pocket ◽  
Structure Relationship ◽  
F Box Protein ◽  
Relationship Of ◽  
Substrate Binding Domain

The SCF ubiquitin ligase comprises four components: Skp1, Cul1, Rbx1 and a variable-subunit F-box protein. The F-box protein Fbs1, which recognizes the N-linked glycoproteins, is involved in the endoplasmic reticulum-associated degradation pathway. Although FBG3, another F-box protein, shares 51% sequence identity with Fbs1, FBG3 does not bind glycoproteins. To investigate the sequence–structure relationship of the substrate-binding pocket, the crystal structure of a mutant substrate-binding domain of Fbs1 in which the six nonconserved regions (β1, β2–β3, β3–β4, β5–β6, β7–β8 and β9–β10) of Fbs1 were substituted with those of FBG3 was determined. The substrate-binding pocket of this model exhibits structural features that differ from those of Fsb1.

CaMKII binds both substrates and effectors at the active site

2020 ◽  
Author(s):  
Can Özden ◽  
Roman Sloutsky ◽  
Nicholas Santos ◽  
Emily Agnello ◽  
Christl Gaubitz ◽  
...  
Keyword(s):  
Active Site ◽  
Specific Binding ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Kinase Domain ◽  
Long Term Memory ◽  
Substrate Binding Pocket ◽  
Interaction Partners ◽  
Dependent Protein

ABSTRACTCa2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein that is required for long-term memory formation. Ca2+/CaM activates CaMKII by binding to its regulatory segment, thereby freeing the substrate binding pocket. One exceptional feature of this kinase is that interaction with specific binding partners persistently activate CaMKII after the Ca2+ stimulus dissipates. The molecular details of this phenomenon are unclear. Despite having a large variety of interaction partners, the specificity of CaMKII has not been structurally well-characterized. We solved X-ray crystal structures of the CaMKII kinase domain bound to four different effectors that modulate CaMKII activity. We show that all four partners use similar interactions to bind across the substrate binding pocket of the CaMKII active site. We generated a sequence alignment based on our structural observations, which revealed conserved interactions. The structures presented here shed much-needed light on CaMKII interactions. These observations will be crucial in guiding further biological experiments.

scholarly journals Molecular mimicry in deoxy-nucleotide catalysis: the structure of Escherichia coli dGTPase reveals the molecular basis of dGTP selectivity: New structural methods offer insight on dGTPases

2018 ◽  
Author(s):  
Christopher O. Barnes ◽  
Ying Wu ◽  
Jinhu Song ◽  
Guowu Lin ◽  
Elizabeth L. Baxter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Reverse Transcription ◽  
Sequence Homology ◽  
Active Site ◽  
Substrate Binding ◽  
Critical Role ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Cellular Survival

AbstractDeoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.Significance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 –which blocks reverse transcription of HIV-1 in macrophages– Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

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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