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1.
Arch Biochem Biophys ; 733: 109471, 2023 01 01.
Article in English | MEDLINE | ID: mdl-36522814

ABSTRACT

NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (H, OH, or CO2-) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.


Subject(s)
Fructose-Bisphosphate Aldolase , Schiff Bases , Fructose-Bisphosphate Aldolase/genetics , Kinetics , Schiff Bases/chemistry , Schiff Bases/metabolism , Lysine , Mutagens , Binding Sites , Aldehyde-Lyases/chemistry , Catalysis , Hydrolases/metabolism , Naphthalenes , Substrate Specificity
2.
Nat Catal ; 5(10): 952-967, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36465553

ABSTRACT

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

3.
Biochemistry ; 2022 May 13.
Article in English | MEDLINE | ID: mdl-35559608

ABSTRACT

The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (∼85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Šresolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues can play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences.

4.
Cell Rep ; 38(2): 110233, 2022 01 11.
Article in English | MEDLINE | ID: mdl-35021089

ABSTRACT

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.


Subject(s)
Leukemia, Myeloid, Acute/metabolism , Phosphoprotein Phosphatases/metabolism , Signal Transduction/physiology , Cell Line, Tumor , Cell Proliferation/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/physiopathology , Phosphoprotein Phosphatases/physiology , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology
5.
Mol Cell ; 81(20): 4147-4164.e7, 2021 10 21.
Article in English | MEDLINE | ID: mdl-34453890

ABSTRACT

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.


Subject(s)
Immunity, Innate , Membrane Proteins/metabolism , Mutation, Missense , Neoplasms/metabolism , Neurofibromin 2/metabolism , Nucleotidyltransferases/metabolism , Tumor Escape , Animals , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Male , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Neurofibromin 2/genetics , Nucleotidyltransferases/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction
7.
Biochemistry ; 60(22): 1776-1786, 2021 06 08.
Article in English | MEDLINE | ID: mdl-34019384

ABSTRACT

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple ß-α-ß structural scaffold. Most members are constructed from a single-core ß-α-ß motif or two consecutively fused ß-α-ß motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.


Subject(s)
Hydrolases/chemistry , Isomerases/chemistry , Amino Acid Sequence , Binding Sites/physiology , Catalysis , Catalytic Domain/physiology , Evolution, Molecular , Kinetics , Magnetic Resonance Spectroscopy/methods , Models, Chemical
8.
PLoS Biol ; 19(2): e3001122, 2021 02.
Article in English | MEDLINE | ID: mdl-33630828

ABSTRACT

The Hippo-YAP pathway responds to diverse environmental cues to manage tissue homeostasis, organ regeneration, tumorigenesis, and immunity. However, how phosphatase(s) directly target Yes-associated protein (YAP) and determine its physiological activity are still inconclusive. Here, we utilized an unbiased phosphatome screening and identified protein phosphatase magnesium-dependent 1A (PPM1A/PP2Cα) as the bona fide and physiological YAP phosphatase. We found that PPM1A was associated with YAP/TAZ in both the cytoplasm and the nucleus to directly eliminate phospho-S127 on YAP, which conferring YAP the nuclear distribution and transcription potency. Accordingly, genetic ablation or depletion of PPM1A in cells, organoids, and mice elicited an enhanced YAP/TAZ cytoplasmic retention and resulted in the diminished cell proliferation, severe gut regeneration defects in colitis, and impeded liver regeneration upon injury. These regeneration defects in murine model were largely rescued via a genetic large tumor suppressor kinase 1 (LATS1) deficiency or the pharmacological inhibition of Hippo-YAP signaling. Therefore, we identify a physiological phosphatase of YAP/TAZ, describe its critical effects in YAP/TAZ cellular distribution, and demonstrate its physiological roles in mammalian organ regeneration.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Protein Phosphatase 2C/metabolism , Regeneration/physiology , Transcription Factors/metabolism , Animals , Cell Proliferation , Cells, Cultured , Colitis/pathology , Humans , Intestines/physiology , Liver Regeneration/physiology , Mice, Inbred C57BL , Mice, Knockout , Organoids , Protein Phosphatase 2C/genetics , Signal Transduction , YAP-Signaling Proteins
9.
J Biol Chem ; 296: 100184, 2021.
Article in English | MEDLINE | ID: mdl-33310704

ABSTRACT

Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.


Subject(s)
HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Magnesium/metabolism , Cations, Divalent/chemistry , Cations, Divalent/metabolism , HIV Infections/virology , HIV Reverse Transcriptase/chemistry , HIV-1/chemistry , HIV-1/metabolism , Humans , Kinetics , Magnesium/chemistry , Molecular Dynamics Simulation , Protein Conformation , Thermodynamics
10.
J Am Chem Soc ; 142(34): 14522-14531, 2020 08 26.
Article in English | MEDLINE | ID: mdl-32623882

ABSTRACT

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation. Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.


Subject(s)
Azo Compounds/chemistry , Carbonic Anhydrases/metabolism , Light , Molecular Probes/chemistry , Sulfonamides/chemistry , Animals , Azo Compounds/chemical synthesis , Carbonic Anhydrases/chemistry , HeLa Cells , Humans , Hydrogen-Ion Concentration , Molecular Docking Simulation , Molecular Probes/chemical synthesis , Molecular Structure , Sulfonamides/chemical synthesis , Zebrafish/embryology , Benzenesulfonamides
11.
ACS Chem Biol ; 15(8): 2259-2272, 2020 08 21.
Article in English | MEDLINE | ID: mdl-32568517

ABSTRACT

The phosphorylation states of RNA polymerase II coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The individual residues of the repetitive heptad of the C-terminal domain (CTD) of the biggest subunit of RNA polymerase II are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles. The mechanism of how the kinases place phosphorylation on the correct serine is not well understood. In this paper, we use biochemical assays, mass spectrometry, molecular modeling, and structural analysis to understand the structural elements determining which serine of the CTD heptad is subject to phosphorylation. We identified three motifs in the activation/P+1 loops differentiating the intrinsic specificity of CTD in various CTD kinases. We characterized the enzyme specificity of the CTD kinases-CDK7 as Ser5-specific, Erk2 with dual specificity for Ser2 and Ser5, and Dyrk1a as a Ser2-specific kinase. We also show that the specificities of kinases are malleable and can be modified by incorporating mutations in their activation/P+1 loops that alter the interactions of the three motifs. Our results provide an important clue to the understanding of post-translational modification of RNA polymerase II temporally during active transcription.


Subject(s)
Protein Kinases/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic , Amino Acid Sequence , Cyclin-Dependent Kinases/metabolism , Discoidin Domain Receptor 1 , Humans , Mass Spectrometry/methods , Phosphorylation , Protein Kinases/chemistry , Protein Processing, Post-Translational , RNA Polymerase II/chemistry , Sequence Homology, Amino Acid , Substrate Specificity , Cyclin-Dependent Kinase-Activating Kinase
12.
Proc Natl Acad Sci U S A ; 117(23): 13000-13011, 2020 06 09.
Article in English | MEDLINE | ID: mdl-32434918

ABSTRACT

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.


Subject(s)
Cystathionine gamma-Lyase/pharmacology , Methionine/antagonists & inhibitors , Mutagenesis, Site-Directed , Prostatic Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Tumor , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/isolation & purification , Cystathionine gamma-Lyase/therapeutic use , DNA Damage/drug effects , Enzyme Assays , Humans , Male , Methionine/blood , Methionine/metabolism , Mice , Poly(ADP-ribose) Polymerases/metabolism , Prostatic Neoplasms/blood , Reactive Oxygen Species/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Toxicity Tests, Acute , Xenograft Model Antitumor Assays
13.
Biochemistry ; 59(16): 1592-1603, 2020 04 28.
Article in English | MEDLINE | ID: mdl-32242662

ABSTRACT

Tautomerase superfamily (TSF) members are constructed from a single ß-α-ß unit or two consecutively joined ß-α-ß units. This pattern prevails throughout the superfamily consisting of more than 11000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4-OT) subgroup and trimers are found in the other four subgroups. One exception is a subset of sequences that are double the length of the short 4-OTs in the 4-OT subgroup, where the coded proteins form trimers. Characterization of two members revealed an interesting dichotomy. One is a symmetric trimer, whereas the other is an asymmetric trimer. One monomer is flipped 180° relative to the other two monomers so that three unique protein-protein interfaces are created that are composed of different residues. A bioinformatics analysis of the fused 4-OT subset shows a further division into two clusters with a total of 133 sequences. The analysis showed that members of one cluster (86 sequences) have more salt bridges if the asymmetric trimer forms, whereas the members of the other cluster (47 sequences) have more salt bridges if the symmetric trimer forms. This hypothesis was examined by the kinetic and structural characterization of two proteins within each cluster. As predicted, all four proteins function as 4-OTs, where two assemble into asymmetric trimers (designated R7 and F6) and two form symmetric trimers (designated W0 and Q0). These findings can be extended to the other sequences in the two clusters in the fused 4-OT subset, thereby annotating their oligomer properties and activities.


Subject(s)
Bacterial Proteins/chemistry , Isomerases/chemistry , Protein Structure, Quaternary , Alcaligenaceae/enzymology , Amino Acid Sequence , Binding Sites , Bordetella/enzymology , Burkholderia/enzymology , Burkholderiaceae/enzymology , Computational Biology , Kinetics , Sequence Alignment
14.
Biochemistry ; 58(22): 2617-2627, 2019 06 04.
Article in English | MEDLINE | ID: mdl-31074977

ABSTRACT

A 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata, and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo- and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF). The TSF has two distinct features: members are composed of a single ß-α-ß unit (homo- and heterohexamer) or two consecutively joined ß-α-ß units (trimer) and generally have a catalytic amino-terminal proline. The enzyme, designated as fused 4-OT, functions as a 4-OT where the active site groups (Pro-1, Arg-39, Arg-76, Phe-115, Arg-127) mirror those in the canonical 4-OT from Pseudomonas putida mt-2. Inactivation by 2-oxo-3-pentynoate suggests that Pro-1 of fused 4-OT has a low p Ka enabling the prolyl nitrogen to function as a general base. A remarkable feature of the fused 4-OT is the absence of P3 rotational symmetry in the structure (1.5 Å resolution). The asymmetric arrangement of the trimer is not due to the fusion of the two ß-α-ß building blocks because an engineered "unfused" variant that breaks the covalent bond between the two units (to generate a heterohexamer) assumes the same asymmetric oligomerization state. It remains unknown how the different active site configurations contribute to the observed overall activities and whether the asymmetry has a biological purpose or role in the evolution of TSF members.


Subject(s)
Bacterial Proteins/chemistry , Isomerases/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Burkholderia/enzymology , Catalytic Domain , Fatty Acids, Unsaturated/chemistry , Isomerases/genetics , Isomerases/isolation & purification , Kinetics , Models, Chemical , Mutation , Protein Structure, Quaternary , Pseudomonas putida/enzymology , Sequence Alignment
15.
ACS Catal ; 9(8): 6955-6961, 2019 Aug 02.
Article in English | MEDLINE | ID: mdl-32257583

ABSTRACT

Ergothioneine is a thiohistidine derivative with potential benefits on many aging-related diseases. The central step of aerobic ergothioneine biosynthesis is the oxidative C-S bond formation reaction catalyzed by mononuclear nonheme iron sulfoxide synthases (EgtB and Egt1). Thus far, only the Mycobacterium thermoresistibile EgtB (EgtB Mth ) crystal structure is available, while the structural information for the more industrially attractive Egt1 enzyme is not. Herein, we reported the crystal structure of the ergothioneine sulfoxide synthase (EgtB Cth ) from Candidatus Chloracidobacterium thermophilum. EgtB Cth has both EgtB- and Egt1-type of activities. Guided by the structural information, we conducted Rosetta Enzyme Design calculations, and we biochemically demonstrated that EgtB Cth can be engineered more toward Egt1-type of activity. This study provides information regarding the factors governing the substrate selectivity in Egt1- and EgtB-catalysis and lays the groundwork for future sulfoxide synthase engineering toward the development of an effective ergothioneine process through a synthetic biology approach.

16.
J Biol Chem ; 293(43): 16851-16861, 2018 10 26.
Article in English | MEDLINE | ID: mdl-30217818

ABSTRACT

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure. REST represses neuronal gene expression in stem cells and non-neuronal cells, but it is minimally expressed in neuronal cells to ensure proper neuronal development. Dysregulation of REST function has been implicated in several cancers and neurological diseases. Modulating REST gene silencing is challenging because cellular and developmental differences can affect its activity. We therefore considered the possibility of modulating REST activity through its regulatory proteins. The human small C-terminal domain phosphatase 1 (SCP1) regulates the phosphorylation state of REST at sites that function as REST degradation checkpoints. Using kinetic analysis and direct visualization with X-ray crystallography, we show that SCP1 dephosphorylates two degron phosphosites of REST with a clear preference for phosphoserine 861 (pSer-861). Furthermore, we show that SCP1 stabilizes REST protein levels, which sustains REST's gene silencing function in HEK293 cells. In summary, our findings strongly suggest that REST is a bona fide substrate for SCP1 in vivo and that SCP1 phosphatase activity protects REST against degradation. These observations indicate that targeting REST via its regulatory protein SCP1 can modulate its activity and alter signaling in this essential developmental pathway.


Subject(s)
Neurons/metabolism , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Amino Acid Motifs , Crystallography, X-Ray , Gene Silencing , HEK293 Cells , Humans , Kinetics , Neurons/chemistry , Nuclear Proteins/genetics , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Stability , Proteolysis , Repressor Proteins/genetics
17.
Methods Enzymol ; 607: 269-297, 2018.
Article in English | MEDLINE | ID: mdl-30149861

ABSTRACT

Proline isomerization is ubiquitous in proteins and is important for regulating important processes such as folding, recognition, and enzymatic activity. In humans, peptidyl-prolyl isomerase cis-trans isomerase NIMA interacting 1 (Pin1) is responsible for mediating fast conversion between cis- and trans-conformations of serine/threonine-proline (S/T-P) motifs in a large number of cellular pathways, many of which are involved in normal development as well as progression of several cancers and diseases. One of the major processes that Pin1 regulates is phosphatase activity against the RNA polymerase II C-terminal domain (RNAPII CTD). However, molecular tools capable of distinguishing the effects of proline conformation on phosphatase function have been lacking. A key tool that allows us to understand isomeric specificity of proteins toward their substrates is the usage of proline mimicking isosteres that are locked to prevent cis/trans-proline conversion. These locked isosteres can be incorporated into standard peptide synthesis and then used in replacement of native substrates in various experimental techniques such as kinetic and thermodynamic assays as well as X-ray crystallography. We will describe the application of these chemical tools in detail using CTD phosphatases as an example. We will also discuss alternative methods for analyzing the effect of proline conformation such as 13C NMR and the biological implications of proline isomeric specificity of proteins. The chemical and analytical tools presented in this chapter are widely applicable and should help elucidate many questions on the role of proline isomerization in biology.


Subject(s)
Enzyme Assays/methods , Peptidylprolyl Isomerase/metabolism , RNA Polymerase II/metabolism , Signal Transduction , Carbon Isotopes/chemistry , Crystallography, X-Ray , Enzyme Assays/instrumentation , Isoenzymes/chemistry , Isoenzymes/metabolism , Isomerism , Models, Molecular , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular , Proline/chemistry , Proline/metabolism , Protein Binding , Protein Domains , RNA Polymerase II/chemistry , Substrate Specificity
18.
Biochemistry ; 57(25): 3524-3536, 2018 06 26.
Article in English | MEDLINE | ID: mdl-29856600

ABSTRACT

NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.


Subject(s)
Aldehyde-Lyases/chemistry , Bacterial Proteins/chemistry , Mycobacterium/enzymology , Oxo-Acid-Lyases/chemistry , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli/metabolism , Models, Molecular , Mycobacterium/chemistry , Mycobacterium/metabolism , Oxo-Acid-Lyases/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Protein Conformation , Protein Multimerization , Sequence Alignment , Substrate Specificity
19.
Cell Chem Biol ; 25(5): 519-529.e4, 2018 05 17.
Article in English | MEDLINE | ID: mdl-29503207

ABSTRACT

Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.


Subject(s)
Ergothioneine/metabolism , Fungal Proteins/metabolism , Lyases/metabolism , Neurospora crassa/metabolism , Biosynthetic Pathways , Catalytic Domain , Crystallography, X-Ray , Fungal Proteins/chemistry , Lyases/chemistry , Neurospora crassa/chemistry , Protein Conformation , Pyridoxal Phosphate/metabolism , Substrate Specificity , Sulfenic Acids/metabolism
20.
Biochemistry ; 57(6): 1012-1021, 2018 Feb 13.
Article in English | MEDLINE | ID: mdl-29303557

ABSTRACT

5-Halo-2-hydroxy-2,4-pentadienoates (5-halo-HPDs) are reportedly generated in the bacterial catabolism of halogenated aromatic hydrocarbons by the meta-fission pathway. The 5-halo-HPDs, where the halogen can be bromide, chloride, or fluoride, result in the irreversible inactivation of 4-oxalocrotonate tautomerase (4-OT), which precedes the enzyme that generates them. The loss of activity is due to the covalent modification of the nucleophilic amino-terminal proline. Mass spectral and crystallographic analysis of the modified enzymes indicates that inactivation of 4-OT by 5-chloro- and 5-bromo-2-hydroxy-2,4-pentadienoate follows a mechanism different from that for the inactivation of 4-OT by 5-fluoro-2-hydroxy-2,4-pentadienoate. The 5-chloro and 5-bromo derivatives undergo 4-OT-catalyzed tautomerization to their respective α,ß-unsaturated ketones followed by attack at C5 (by the prolyl nitrogen) with concomitant loss of the halide. For the 5-fluoro species, the presence of a small amount of the α,ß-unsaturated ketone could result in a Michael addition of the prolyl nitrogen to C4 followed by protonation at C3. The fluoride is not eliminated. These observations suggest that the inactivation of 4-OT by a downstream metabolite could hamper the efficacy of the pathway, which is the first time that such a bottleneck has been reported for the meta-fission pathway.


Subject(s)
Fatty Acids, Unsaturated/metabolism , Isomerases/metabolism , Pseudomonas putida/enzymology , Crystallography, X-Ray , Enzyme Activation , Fatty Acids, Unsaturated/chemistry , Halogenation , Isomerases/chemistry , Kinetics , Models, Molecular , Pseudomonas putida/chemistry , Pseudomonas putida/metabolism
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