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1.
Biochemistry ; 2024 Feb 02.
Article in English | MEDLINE | ID: mdl-38306231

ABSTRACT

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

2.
PLoS One ; 16(4): e0248991, 2021.
Article in English | MEDLINE | ID: mdl-33857156

ABSTRACT

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.


Subject(s)
Escherichia coli Proteins/chemistry , Ureohydrolases/chemistry , Catalytic Domain , Conserved Sequence , Escherichia coli , Escherichia coli Proteins/metabolism , Ureohydrolases/metabolism
3.
Photochem Photobiol Sci ; 20(2): 255-263, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33721251

ABSTRACT

The photochemistry of Fe(III) coordinated to natural uronate-containing polysaccharides has been investigated quantitatively in aqueous solution. It is demonstrated that the photoreduction of the coordinated Fe(III) to Fe(II) and oxidative decarboxylation occurs in a variety of uronate-containing polysaccharides. The photochemistry of the Fe(III)-polyuronic acid system generated a radical species during the reaction which was studied using the spin trapping technique. The identity of the radical species from this reaction was confirmed as CO2•- indicating that both bond cleavage of the carboxylate and oxidative decarboxylation after ligand to metal charge transfer radical reactions may be taking place upon irradiation. Degradation of the polyuronic acid chain was investigated with dynamic light scattering, showing a decrease in the hydrodynamic radius of the polymer assemblies in solution after light irradiation that correlates with the Fe(II) generation. A decrease in viscosity of Fe(IIII)-alginate after light irradiation was also observed. Additionally, the photochemical reaction was investigated in plant root tissue (parsnip) demonstrating that Fe(III) coordination in these natural materials leads to photoreactivity that degrades the pectin component. These results highlight that this Fe(III)-polyuronic acid can occur in many natural systems and may play a role in biogeochemical cycling of iron and ferrous iron generation in plants with significant polyuronic acid content.

4.
Science ; 359(6381): 1247-1250, 2018 03 16.
Article in English | MEDLINE | ID: mdl-29590073

ABSTRACT

Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme.


Subject(s)
Archaeal Proteins/chemistry , Histidine/analogs & derivatives , Iron-Sulfur Proteins/chemistry , Pyrococcus horikoshii/enzymology , S-Adenosylmethionine/chemistry , Carbon/chemistry , Crystallography, X-Ray , Histidine/biosynthesis , Iron/chemistry , Organometallic Compounds/chemistry
5.
ACS Chem Biol ; 13(3): 591-599, 2018 03 16.
Article in English | MEDLINE | ID: mdl-29210568

ABSTRACT

IscU, the central scaffold protein in the bacterial ISC iron-sulfur (Fe-S) cluster biosynthesis system, has long been recognized to bind a Zn2+ ion at its active site. While initially regarded as an artifact, Zn2+ binding has been shown to induce stabilization of the IscU structure that may mimic a state biologically relevant to IscU's role in Fe-S cluster biosynthesis. More recent studies have revealed that SufU, a homologous protein involved in Fe-S cluster biosynthesis in Gram-positive bacteria, also binds a Zn2+ ion with structural implications. Given the widespread occurrence of the "IscU-like" protein fold, particularly among Fe-S cluster biosynthesis systems, an interesting question arises as to whether Zn2+ ion binding and the resulting structural alterations are common properties in IscU-like proteins. Interactions between IscU and specific metal ions are investigated and compared side-by-side with those of SufU from a representative Gram-positive bacterium in the phylum Firmicutes. These studies were extended with additional transition metal ions chosen to investigate the influence of coordination geometry on selectivity for binding at the active sites of IscU and SufU. Monitoring and comparing the conformational behavior and stabilization afforded by different transition metal ions upon IscU and SufU revealed similarities between the two proteins and suggest that metal-dependent conformational transitions may be characteristic of U-type proteins involved in Fe-S cluster biosynthesis.


Subject(s)
Bacterial Proteins/drug effects , Escherichia coli Proteins/drug effects , Iron-Sulfur Proteins/drug effects , Lyases/drug effects , Transition Elements/pharmacology , Zinc/pharmacology , Bacterial Proteins/chemistry , Catalytic Domain , Cations , Iron-Sulfur Proteins/chemistry , Protein Binding , Protein Conformation/drug effects
6.
Mol Biosyst ; 7(1): 74-81, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20931132

ABSTRACT

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.


Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Histidine/analogs & derivatives , Iron-Sulfur Proteins/metabolism , Pyrococcus horikoshii/enzymology , Pyrococcus horikoshii/metabolism , Archaeal Proteins/genetics , Chromatography, Liquid , Electron Spin Resonance Spectroscopy , Histidine/biosynthesis , Histidine/chemistry , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Mass Spectrometry , Molecular Structure , Mutation , Protein Multimerization
7.
Nature ; 465(7300): 891-6, 2010 Jun 17.
Article in English | MEDLINE | ID: mdl-20559380

ABSTRACT

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the C(gamma,Met)-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry.


Subject(s)
Archaeal Proteins/metabolism , Free Radicals/metabolism , Histidine/analogs & derivatives , Iron-Sulfur Proteins/metabolism , Pyrococcus horikoshii/enzymology , Free Radicals/chemistry , Histidine/biosynthesis , Histidine/chemistry , S-Adenosylmethionine/metabolism
8.
J Biol Chem ; 284(17): 11012-6, 2009 Apr 24.
Article in English | MEDLINE | ID: mdl-19261617

ABSTRACT

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ(1) riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ(1) aptamer domain in complex with its precursor metabolite preQ(0). The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated.


Subject(s)
Aptamers, Nucleotide/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , Bacillus subtilis/metabolism , Base Pairing , Binding Sites , Crystallography, X-Ray/methods , Electrons , Hydrogen Bonding , Ligands , Models, Chemical , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Protein Structure, Tertiary , Thermoanaerobacter/metabolism
9.
Biochem Biophys Res Commun ; 371(1): 154-8, 2008 Jun 20.
Article in English | MEDLINE | ID: mdl-18423397

ABSTRACT

Reaction-intermediate analogs have been used to understand how phosphoryl transfer enzymes promote catalysis. Herein we report the first structure of a small ribozyme crystallized with a 3'-OH, 2',5'-linkage in lieu of the normal phosphodiester substrate. The new structure shares features of the reaction coordinate exhibited in prior ribozyme structures including a vanadate complex that mimicked the oxyphosphorane transition state. As such, the structure exhibits reaction-intermediate traits that allow direct comparison of stabilizing interactions to the 3'-OH, 2',5'-linkage contributed by the RNA enzyme and its protein counterpart, ribonuclease. Clear similarities are observed between the respective structures including hydrogen bonds to the non-bridging oxygens of the scissile phosphate. Other commonalities include carefully poised water molecules that may alleviate charge build-up in the transition state and placement of a positive charge near the leaving group. The advantages of 2',5'-linkages to investigate phosphoryl-transfer reactions are discussed, and argue for their expanded use in structural studies.


Subject(s)
RNA, Catalytic/chemistry , Ribonucleases/chemistry , Binding Sites , Catalysis , Crystallization , Nucleic Acid Conformation , Protein Conformation , X-Ray Diffraction
10.
RNA ; 13(7): 1052-70, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17488874

ABSTRACT

The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 A resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2',5'-phosphodiester linkage at the site of cleavage was also solved at 2.35 A resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2',5' structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2'-O nucleophile with the scissile G+1 phosphorus. The 2',5'-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2',5' linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.


Subject(s)
Phase Transition/drug effects , RNA Stability/drug effects , RNA, Catalytic/chemistry , Vanadates/metabolism , Water/pharmacology , Base Sequence , Binding Sites , Catalysis/drug effects , Crystallography, X-Ray , Models, Biological , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Catalytic/drug effects , RNA, Catalytic/metabolism , Vanadates/chemistry , Water/chemistry
11.
J Immunol ; 177(1): 355-61, 2006 Jul 01.
Article in English | MEDLINE | ID: mdl-16785531

ABSTRACT

In mammals, activation-induced deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes. SHM and CSR activities require separate regions within AID. A chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) at the AID C terminus is necessary for CSR, and has been suggested to associate with CSR-specific cofactors. CSR appeared late in AID evolution, during the emergence of land vertebrates from bony fish, which only display SHM. Here, we show that AID from African clawed frog (Xenopus laevis), but not pufferfish (Takifugu rubripes), can induce CSR in AID-deficient mouse B cells, although both are catalytically active in bacteria and mammalian cell systems, albeit at decreased level. Like mammalian AID, Takifugu AID is actively exported from the cell nucleus by CRM1, and the Takifugu NES can substitute for the equivalent region in human AID, indicating that all the CSR-essential NES motif functions evolutionarily predated CSR activity. We also show that fusion of the Takifugu AID catalytic domain to the entire human noncatalytic domain restores activity in mammalian cells, suggesting that AID features mapping within the noncatalytic domain, but outside the NES, influence its function.


Subject(s)
Cytidine Deaminase/chemistry , Cytidine Deaminase/physiology , Phylogeny , Amino Acid Sequence , Animals , Catalytic Domain/genetics , Cell Line , Cloning, Molecular , Cytidine Deaminase/deficiency , Cytidine Deaminase/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , Immunoglobulin Class Switching/genetics , Mice , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Nuclear Export Signals/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Sequence Homology, Amino Acid , Takifugu , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus Proteins/physiology , Xenopus laevis
12.
Proc Natl Acad Sci U S A ; 101(21): 8114-9, 2004 May 25.
Article in English | MEDLINE | ID: mdl-15148397

ABSTRACT

Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-A resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site "flap" whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.


Subject(s)
Cytidine Deaminase/chemistry , Cytidine Deaminase/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , APOBEC-1 Deaminase , Binding Sites , Crystallography, X-Ray , Humans , Immunoglobulin M/immunology , Models, Molecular , Mutation , Protein Conformation , RNA Editing , Syndrome
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