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Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase (ESCO1)
548a
Wednesday, March 2, 2016
from a molecular docking campaign on several libraries. We are currently testing in vitro the most promising compounds from this in silico screening to identify high affinity hits able to inhibit human APT1. 2701-Pos Board B78 Structural and Biochemical Investigations on the Catalytic Mechanism of Pyridoxal Kinase (PdxK) from Salmonella Typhimurium and its Interactions with PLP-Dependent Enzymes G. Deka1, J.F. Benazir1, J.N. Kalyani2, H.S. Savithri2, M.R.N. Murthy1. 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, 2 Biochemistry Department, Indian Institute of Science, Bangalore, India. Pyridoxal kinase (PdxK), a member of ribokinase superfamily of enzymes, is involved in pyridoxal 5’ phosphate (PLP) synthesis by a salvage pathway. PdxK ensures availability of PLP to a large number of enzymes involved in amino acid and sugar metabolism that use PLP as a cofactor and is regarded as a potential drug target. We have determined the crystal structure of PdxK from Salmonella typhimurium (stPLK) in its unliganded form as well ˚ , 1.9A ˚ and 2.5A ˚ resas in complex with Mg ATP and Mg ATP-PLP at 2.6A olutions, respectively. The protomeric structure of the dimeric enzyme consisting of three layered aba structure is similar to those of other ribokinase family proteins. A segment of residues 134-142 constituting a flexible loop undergoes a large conformational change from an open form to a closed form upon ligand binding and guards the active site from solvent exposure and prevents premature hydrolysis of ATP. During catalysis, the substrate ˚ to interact with the g phosphate of ATP bound PL moves by a distance of ~6A near an anion hole constituted by residues 234-237 (GTGD). As reported for the E. coli enzyme, kinetic studies show that stPLK has higher activity in the presence of Mg2þ when compared to other divalent metal ions. High concentration of PLP was found to inhibit stPLK and the structure of crystals obtained in presence of excess PLP reveals that PLP is covalently attached as an internal aldimine to Lys233. Surface Plasmon resonance (SPR ) and ELISA studies show that stPLK specifically interacts with diaminopropionate ammonia lyase, a fold type II PLP dependent enzyme, suggesting probable direct transfer of the product PLP from PdxK to the apo form of PLPdependent enzymes. 2702-Pos Board B79 Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase (ESCO1) Yadilette Rivera-Colon, Andrew Maguire, Glen P. Liszczak, Ronen Marmorstein. Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA. Cell division in both prokaryotes and eukaryotes relies on the accurate segregation of chromosomes. The Structural Maintenance of Chromosomes (SMC)heterodimeric ATPase protein complex,cohesin, plays a key role in this process as it is responsible for cohesion between sister chromatids and is a major constituent of interphase and mitotic chromosomes. The functions of cohesin are modulated by phosphorylation, acetylation, ATP hydrolysis, and site-specific proteolysis of its SMC1 and/or SMC3 subunits. Establishment of sister chromatid cohesion is mediated by acetylation of the cohesin subunit SMC3 by the lysine acetyltransferase ESCO1. ESCO2 is a paralogous human cohesin acetyltransferase and genetic defects that reduce ESCO2 activity lead to Roberts Syndrome, a childhood autosomal recessive disorder that manifests by mental retardation, craniofacial abnormalities and limb reduction. We have determined the X-ray crystal structure of ESCO1 and have carried out structure-based mutagenesis, biochemical and enzymatic studies. Together, these studies provide novel insights into SMC3-specific acetylation by ESCO1 and rationalize the functional consequence of ESCO2 mutations correlated with Roberts Syndrome. 2703-Pos Board B80 Origins of Catalytic Specificity in Bacterial Oligosaccharyltransferase Brittany R. Morgan, Francesca Massi. Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. Modification of polypeptides through asparagine (N)-linked glycosylation is involved in a broad range of biological functions. N-linked glycosylation is catalyzed by oligosaccharyltransferase (OST), an endoplasmic reticulum membrane protein, which promotes the formation of an N-glycosidic linkage between the acceptor asparagine and an oligosaccharide donor. Broad polypeptide substrate specificity is characteristic of N-linked glycosylation, resulting from a short consensus sequence: N-X-S/T (where X is any amino acid except P). However, experimental evidence has shown that the N-glycosylation efficiency is affected by the sequence within this consensus motif.
We investigated the catalytic specificity of Campylobacter lari OST using molecular dynamics simulations. In OST, a large external loop (EL5) pins the substrate in the binding pocket located between the transmembrane and periplasmic domains while two acidic catalytic residues, E319 and D56 in C. lari, are in position to form hydrogen bonds with the acceptor asparagine and prime it for nucleophilic attack on the oligosaccharide donor. To explore how different substrates affect the efficiency of N-linked glycosylation, four substrates of differing N-glycosylation efficiencies were examined: the optimal consensus sequence (NAT) and three sub-optimal variants (NAS, NFS, and NWS). Our simulations of OST in complex with the optimal substrate (NAT) show a conformational change of EL5 and of the periplasmic domain is necessary to promote optimal hydrogen bond formation between the acceptor asparagine and E319/D56 by restricting side chain motion in the catalytic pocket. In addition, we found that binding of the other three substrates (NAS, NFS, and NWS) affects the structure and dynamics of OST. These changes lower the probability of forming hydrogen bonds with the acceptor asparagine, essential for catalysis, and accelerate substrate release for NFS, providing two mechanisms for modulating the glycosylation efficiency of OST for various consensus sequence substrates. 2704-Pos Board B81 Molecular Mechanism of the Catalytic Reaction of no Reductase Revealed by Novel Time-Resolved Visible/IR Absorption Spectrometers with Microfluidic Device Tetsunari Kimura1,2, Hanae Takeda1,3, Shoko Ishii1,3, Takehiko Tosha1, Yoshitsugu Shiro1,3, Minoru Kubo1,4. 1 SPring-8 Ctr., RIKEN, Sayo, Japan, 2Grad. Sch. Sci., Kobe Univ., Kobe, Japan, 3Grad. Sch. Life Sci., Univ. of Hyogo, Kamigori, Japan, 4Presto, JST, Kawaguchi, Japan. Time-resolved (TR) spectroscopy plays convincing roles in clarifying the molecular mechanism of biological reactions in the atomic and electronic level. Most of the biological reactions can be triggered by the sudden changes in buffer conditions, but the time-resolution of the conventional solution-mixing technique is limited to several milliseconds and the sample consumption is enormous, resulting in the limited applications of TR spectroscopy. Here, to investigate the enzymatic reaction of a low-yield membrane protein with microsecond-resolution, novel flow-flash TR-visible/IR spectrometers were developed. Time-resolution of microseconds was achieved using cagedcompounds, which release substrates upon laser flash. Combinational use of a micro-channel flow-cell and a nano-liter step-pulse syringe-pump synchronized with the microscopic laser flashes realized the spectral accumulation with the minimal sample consumption. The developed system was applied to nitric-oxide reductase (NOR), a membrane enzyme that catalyzes NO reduction (2NO þ 2Hþ þ 2e- -> N2O þ H2O) in the bacterium denitrification process. Although our X-ray crystallographic analysis has revealed the atomic structure of catalytic center consisting of heme b3 and non-heme FeB, the molecular mechanism of NO reduction is still controversial. This is due to the difficulties in direct observation of the transient NO-bound form, whose lifetime is shorter than 1 ms. Our newly developed TR-visible absorption spectrometer, which probed the electronic state of heme b3, revealed that NO bound to heme b3 within 4 ms and was reduced with a time constant of 100 ms. TR-IR measurement at 10 ms showed that another NO molecule bound to FeB. These TR measurements revealed that each iron in the active center binds different NO molecule in the early stage of the reaction and the subsequent N-N bond formation occurs in the intramolecular manner. 2705-Pos Board B82 Droplet-Based Microfluidics for Measuring Enzymatic Activities: Application to L-Asparaginase used in Antileukemic Therapy Manfred W. Konrad1, Christos S. Karamitros1, Joanan Lopez Morales1, Jean-Christophe Baret2. 1 Enzyme Biochemistry, Max Planck Institute, Goettingen, Germany, 2Soft MicroSystems, CRPP, CNRS, University of Bordeaux, Bordeaux, France. Our work aims to ameliorate catalytic properties of L-asparaginase (L-ASNase) which is a protein drug used in antileukemic therapy. Bacterial L-ASNases are FDA-approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn. Their therapeutic efficacy is based on the fact that several hematological malignancies, in particular Acute Lymphoblastic Leukemia (ALL), depend for growth on the extracellular supply of L-Asn. To avoid various side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. In order to find variants of improved specific activities in mutated enzyme libraries, we developed a droplet-based microfluidic platform for high throughput and miniaturization of kinetic assays that can be performed not only on purified
Wednesday, March 2, 2016
from a molecular docking campaign on several libraries. We are currently testing in vitro the most promising compounds from this in silico screening to identify high affinity hits able to inhibit human APT1. 2701-Pos Board B78 Structural and Biochemical Investigations on the Catalytic Mechanism of Pyridoxal Kinase (PdxK) from Salmonella Typhimurium and its Interactions with PLP-Dependent Enzymes G. Deka1, J.F. Benazir1, J.N. Kalyani2, H.S. Savithri2, M.R.N. Murthy1. 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India, 2 Biochemistry Department, Indian Institute of Science, Bangalore, India. Pyridoxal kinase (PdxK), a member of ribokinase superfamily of enzymes, is involved in pyridoxal 5’ phosphate (PLP) synthesis by a salvage pathway. PdxK ensures availability of PLP to a large number of enzymes involved in amino acid and sugar metabolism that use PLP as a cofactor and is regarded as a potential drug target. We have determined the crystal structure of PdxK from Salmonella typhimurium (stPLK) in its unliganded form as well ˚ , 1.9A ˚ and 2.5A ˚ resas in complex with Mg ATP and Mg ATP-PLP at 2.6A olutions, respectively. The protomeric structure of the dimeric enzyme consisting of three layered aba structure is similar to those of other ribokinase family proteins. A segment of residues 134-142 constituting a flexible loop undergoes a large conformational change from an open form to a closed form upon ligand binding and guards the active site from solvent exposure and prevents premature hydrolysis of ATP. During catalysis, the substrate ˚ to interact with the g phosphate of ATP bound PL moves by a distance of ~6A near an anion hole constituted by residues 234-237 (GTGD). As reported for the E. coli enzyme, kinetic studies show that stPLK has higher activity in the presence of Mg2þ when compared to other divalent metal ions. High concentration of PLP was found to inhibit stPLK and the structure of crystals obtained in presence of excess PLP reveals that PLP is covalently attached as an internal aldimine to Lys233. Surface Plasmon resonance (SPR ) and ELISA studies show that stPLK specifically interacts with diaminopropionate ammonia lyase, a fold type II PLP dependent enzyme, suggesting probable direct transfer of the product PLP from PdxK to the apo form of PLPdependent enzymes. 2702-Pos Board B79 Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase (ESCO1) Yadilette Rivera-Colon, Andrew Maguire, Glen P. Liszczak, Ronen Marmorstein. Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA. Cell division in both prokaryotes and eukaryotes relies on the accurate segregation of chromosomes. The Structural Maintenance of Chromosomes (SMC)heterodimeric ATPase protein complex,cohesin, plays a key role in this process as it is responsible for cohesion between sister chromatids and is a major constituent of interphase and mitotic chromosomes. The functions of cohesin are modulated by phosphorylation, acetylation, ATP hydrolysis, and site-specific proteolysis of its SMC1 and/or SMC3 subunits. Establishment of sister chromatid cohesion is mediated by acetylation of the cohesin subunit SMC3 by the lysine acetyltransferase ESCO1. ESCO2 is a paralogous human cohesin acetyltransferase and genetic defects that reduce ESCO2 activity lead to Roberts Syndrome, a childhood autosomal recessive disorder that manifests by mental retardation, craniofacial abnormalities and limb reduction. We have determined the X-ray crystal structure of ESCO1 and have carried out structure-based mutagenesis, biochemical and enzymatic studies. Together, these studies provide novel insights into SMC3-specific acetylation by ESCO1 and rationalize the functional consequence of ESCO2 mutations correlated with Roberts Syndrome. 2703-Pos Board B80 Origins of Catalytic Specificity in Bacterial Oligosaccharyltransferase Brittany R. Morgan, Francesca Massi. Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. Modification of polypeptides through asparagine (N)-linked glycosylation is involved in a broad range of biological functions. N-linked glycosylation is catalyzed by oligosaccharyltransferase (OST), an endoplasmic reticulum membrane protein, which promotes the formation of an N-glycosidic linkage between the acceptor asparagine and an oligosaccharide donor. Broad polypeptide substrate specificity is characteristic of N-linked glycosylation, resulting from a short consensus sequence: N-X-S/T (where X is any amino acid except P). However, experimental evidence has shown that the N-glycosylation efficiency is affected by the sequence within this consensus motif.
We investigated the catalytic specificity of Campylobacter lari OST using molecular dynamics simulations. In OST, a large external loop (EL5) pins the substrate in the binding pocket located between the transmembrane and periplasmic domains while two acidic catalytic residues, E319 and D56 in C. lari, are in position to form hydrogen bonds with the acceptor asparagine and prime it for nucleophilic attack on the oligosaccharide donor. To explore how different substrates affect the efficiency of N-linked glycosylation, four substrates of differing N-glycosylation efficiencies were examined: the optimal consensus sequence (NAT) and three sub-optimal variants (NAS, NFS, and NWS). Our simulations of OST in complex with the optimal substrate (NAT) show a conformational change of EL5 and of the periplasmic domain is necessary to promote optimal hydrogen bond formation between the acceptor asparagine and E319/D56 by restricting side chain motion in the catalytic pocket. In addition, we found that binding of the other three substrates (NAS, NFS, and NWS) affects the structure and dynamics of OST. These changes lower the probability of forming hydrogen bonds with the acceptor asparagine, essential for catalysis, and accelerate substrate release for NFS, providing two mechanisms for modulating the glycosylation efficiency of OST for various consensus sequence substrates. 2704-Pos Board B81 Molecular Mechanism of the Catalytic Reaction of no Reductase Revealed by Novel Time-Resolved Visible/IR Absorption Spectrometers with Microfluidic Device Tetsunari Kimura1,2, Hanae Takeda1,3, Shoko Ishii1,3, Takehiko Tosha1, Yoshitsugu Shiro1,3, Minoru Kubo1,4. 1 SPring-8 Ctr., RIKEN, Sayo, Japan, 2Grad. Sch. Sci., Kobe Univ., Kobe, Japan, 3Grad. Sch. Life Sci., Univ. of Hyogo, Kamigori, Japan, 4Presto, JST, Kawaguchi, Japan. Time-resolved (TR) spectroscopy plays convincing roles in clarifying the molecular mechanism of biological reactions in the atomic and electronic level. Most of the biological reactions can be triggered by the sudden changes in buffer conditions, but the time-resolution of the conventional solution-mixing technique is limited to several milliseconds and the sample consumption is enormous, resulting in the limited applications of TR spectroscopy. Here, to investigate the enzymatic reaction of a low-yield membrane protein with microsecond-resolution, novel flow-flash TR-visible/IR spectrometers were developed. Time-resolution of microseconds was achieved using cagedcompounds, which release substrates upon laser flash. Combinational use of a micro-channel flow-cell and a nano-liter step-pulse syringe-pump synchronized with the microscopic laser flashes realized the spectral accumulation with the minimal sample consumption. The developed system was applied to nitric-oxide reductase (NOR), a membrane enzyme that catalyzes NO reduction (2NO þ 2Hþ þ 2e- -> N2O þ H2O) in the bacterium denitrification process. Although our X-ray crystallographic analysis has revealed the atomic structure of catalytic center consisting of heme b3 and non-heme FeB, the molecular mechanism of NO reduction is still controversial. This is due to the difficulties in direct observation of the transient NO-bound form, whose lifetime is shorter than 1 ms. Our newly developed TR-visible absorption spectrometer, which probed the electronic state of heme b3, revealed that NO bound to heme b3 within 4 ms and was reduced with a time constant of 100 ms. TR-IR measurement at 10 ms showed that another NO molecule bound to FeB. These TR measurements revealed that each iron in the active center binds different NO molecule in the early stage of the reaction and the subsequent N-N bond formation occurs in the intramolecular manner. 2705-Pos Board B82 Droplet-Based Microfluidics for Measuring Enzymatic Activities: Application to L-Asparaginase used in Antileukemic Therapy Manfred W. Konrad1, Christos S. Karamitros1, Joanan Lopez Morales1, Jean-Christophe Baret2. 1 Enzyme Biochemistry, Max Planck Institute, Goettingen, Germany, 2Soft MicroSystems, CRPP, CNRS, University of Bordeaux, Bordeaux, France. Our work aims to ameliorate catalytic properties of L-asparaginase (L-ASNase) which is a protein drug used in antileukemic therapy. Bacterial L-ASNases are FDA-approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn. Their therapeutic efficacy is based on the fact that several hematological malignancies, in particular Acute Lymphoblastic Leukemia (ALL), depend for growth on the extracellular supply of L-Asn. To avoid various side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. In order to find variants of improved specific activities in mutated enzyme libraries, we developed a droplet-based microfluidic platform for high throughput and miniaturization of kinetic assays that can be performed not only on purified