Molecular Engineering of E. coli Bacterioferritin: A Versatile Nanodimensional Protein Cage.

Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering.

Squarate-based carbocyclic nucleosides: Syntheses, computational analyses and anticancer/antiviral evaluation.

Squaric acid and its derivatives are versatile synthons and have demonstrated applications in medicinal chemistry, notably as non-classical bioisosteric replacements for functional groups such as carboxylic acids, alpha-amino acids, urea, guanidine, peptide bonds and phosphate/pyrophosphate linkages. Surprisingly, no reports have appeared concerning its possible application as a nucleobase substitute in nucleosides. A preliminary investigation of such an application is reported herein. 3-Amino-4-((1R,4S)-4-(hydroxymethyl)cyclopent-2-en-1-yl)amino-cyclobut-3-ene-1,2-dione, 3-((1R,4S)-4-(hydroxymethyl)cyclopent-2-en-1-yl)amino-4-methoxycyclobut-3-ene-1,2-dione, and 3-hydroxy-4-((1R,4S)-4-(hydroxymethyl)cyclopent-2-en-1-yl)amino-cyclobut-3-ene-1,2-dione sodium salt were synthesized. Computational analyses of their structures and preliminary antitumor and antiviral screening results are reported.

The crystal structure of a homodimeric Pseudomonas glyoxalase†I enzyme reveals asymmetric metallation commensurate with half-of-sites activity.

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni(2+) ion, whereas the other contains two inactivating Zn(2+) ions. Enzymological experiments prompted by the binuclear Zn(2+) site identified a novel catalytic property of GloA2. The enzyme can function as a Zn(2+) /Co(2+) -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.

Molecular interactions between thiostrepton and the TipAS protein from Streptomyces lividans.

In Streptomyces lividans, the expression of several proteins is stimulated by the thiopeptide antibiotic thiostrepton. Two of these, TipAL and TipAS, autoregulate their expression after covalently binding to thiostrepton; this irreversibly sequesters the antibiotic and desensitizes the organism to its effects. In this work, additional molecular recognition interactions involved in this critical event were explored by utilizing various thiostrepton analogues and several site-directed mutants of the TipAS antibiotic binding protein. Dissociation constants for several thiostrepton analogues ranged from 0.19 to 12.95 µM, depending on the analogue. The contributions of specific structural elements of the thiostrepton molecule to this interaction have been discerned, and an unusual covalent modification between the antibiotic and a new residue in a TipAS mutant has been detected.

Bacterial glyoxalase I enzymes: structural and biochemical investigations.

A number of bacterial glyoxalase I enzymes are maximally activated by Ni2+ and Co2+ ions, but are inactive in the presence of Zn2+, yet these enzymes will also bind this metal ion. The structure-activity relationships between these two classes of glyoxalase I serve as important clues as to how the molecular structures of these proteins control metal-activation profiles.

Oxocarbon Acids and their Derivatives in Biological and Medicinal Chemistry.

The biological and medicinal chemistry of the oxocarbon acids 2,3- dihydroxycycloprop-2-en-1-one (deltic acid), 3,4-dihydroxycyclobut-3-ene-1,2-dione (squaric acid), 4,5-dihydroxy-4-cyclopentene-1,2,3-trione (croconic acid), 5,6-dihydroxycyclohex- 5-ene-1,2,3,4-tetrone (rhodizonic acid) and their derivatives is reviewed and their key chemical properties and reactions are discussed. Applications of these compounds as potential bioisosteres in biological and medicinal chemistry are examined. Reviewed areas include cell imaging, bioconjugation reactions, antiviral, antibacterial, anticancer, enzyme inhibition, and receptor pharmacology.

Bacterial glyoxalase enzymes.

The glyoxalase system is composed of two metalloenzymes, Glyoxalase I and Glyoxalase II. This system is important in the detoxification of methylglyoxal, among other roles. Detailed studies have determined that a number of bacterial Glyoxalase I enzymes are maximally activated by Ni(2+) and Co(2+) ions, but are inactive in the presence of Zn(2+). This is in contrast to the Glyoxalase I enzyme from humans, which is catalytically active with Zn(2+) as well as a number of other metal ions. The structure-activity relationships between these two classes of Glyoxalase I are serving as important clues to how the molecular structures of these proteins control metal activation profiles as well as to clarify the mechanistic chemistry of these catalysts. In addition, the possibility of targeting inhibitors against the bacterial versus human enzyme has the potential to lead to new approaches to combat bacterial infections.

Structural variation in bacterial glyoxalase I enzymes: investigation of the metalloenzyme glyoxalase I from Clostridium acetobutylicum.

The glyoxalase system catalyzes the conversion of toxic, metabolically produced α-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn(2+) and non-Zn(2+) activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni(2+)/Co(2+)-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn(2+)-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.

Mechanistic studies on the enzymatic processing of fluorinated methionine analogues by Trichomonas vaginalis methionine γ-lyase.

TFM (L-trifluoromethionine), a potential prodrug, was reported to be toxic towards human pathogens that express MGL (L-methionine γ-lyase; EC 4.4.1.11), a pyridoxal phosphate-containing enzyme that converts L-methionine into α-oxobutyrate, ammonia and methyl mercaptan. It has been hypothesized that the extremely reactive thiocarbonyl difluoride is produced when the enzyme acts upon TFM, resulting in cellular toxicity. The potential application of the fluorinated thiomethyl group in other areas of biochemistry and medicinal chemistry requires additional studies. Therefore a detailed investigation of the theoretical and experimental chemistry and biochemistry of these fluorinated groups (CF3S⁻ and CF2HS⁻) has been undertaken to trap and identify chemical intermediates produced by enzyme processing of molecules containing these fluorinated moieties. TvMGL (MGL from Trichomonas vaginalis) and a chemical model system of the reaction were utilized in order to investigate the cofactor-dependent activation of TFM and previously uninvestigated DFM (L-difluoromethionine). The differences in toxicity between TFM and DFM were evaluated against Escherichia coli expressing TvMGL1, as well as the intact human pathogen T. vaginalis. The relationship between the chemical structure of the reactive intermediates produced from the enzymatic processing of these analogues and their cellular toxicity are discussed.

Imparting conformational memory for material adhesion.

Adhesion between similar and dissimilar materials is essential to many biological systems and synthetic materials, devices, and machines. Since the inception of adhesion science more than five decades ago, adhesion to a surface has long been recognized as beyond two-dimensional. Similarly, molecular conformation - the three-dimensional arrangement of atoms in a molecule - is ubiquitous in biology and fundamental to the binding of biomolecules. However, the connection between these concepts, which could link molecular conformation in biology to micro- and macroscopic adhesion in materials science, remains elusive. Herein, we examine this connection by manipulating the molecular conformation of a mussel-inspired universal coating, which imparts a memory for recognizing different hydrogels. This approach leads to significantly (several fold) increased interfacial adhesion between the coating and hydrogels across a broad range of length scales, from molecular to macroscopic. Furthermore, we demonstrate that imparting memory is a general and facile noncovalent approach for enhancing interfacial adhesion that, with suitable energy dissipation, can be used for the bonding of materials.

Controllable delivery of small-molecule compounds to targeted cells utilizing carbon nanotubes.

Carbon nanotubes (CNTs) have emerged as a new alternative and efficient tool for transporting molecules with biotechnological and biomedical applications, because of their remarkable physicochemical properties. Encapsulation of functional molecules into the hollow chambers of CNTs can not only stabilize encapsulated molecules but also generate new nanodevices. In this work, we have demonstrated that CNTs can function as controllable carriers to transport small-molecule compounds (SMCs) loaded inside their hollow tunnels onto targeted cells. Using indole as model compound, CNTs can protect indole molecules during transportation. Labeling indole-loaded CNTs (indole@CNTs) with EphB4-binding peptides generates cell-homing indole@CNTs (CIDs). CIDs can selectively target EphB4-expressing cells and release indole onto cell surfaces by near-infrared (NIR) irradiation. Released indole molecules exhibit significant cell-killing effects without causing local overheating. This establishes CNTs as excellent near-infrared controllable delivery vehicles for SMCs as selective cell-killing agents.

The Charge-State and Structural Stability of Peptides Conferred by Microsolvating Environments in Differential Mobility Spectrometry.

The presence of solvent vapor in a differential mobility spectrometry (DMS) cell creates a microsolvating environment that can mitigate complications associated with field-induced heating. In the case of peptides, the microsolvation of protonation sites results in a stabilization of charge density through localized solvent clustering, sheltering the ion from collisional activation. Seeding the DMS carrier gas (N2) with a solvent vapor prevented nearly all field-induced fragmentation of the protonated peptides GGG, AAA, and the Lys-rich Polybia-MP1 (IDWKKLLDAAKQIL-NH2). Modeling the microsolvation propensity of protonated n-propylamine [PrNH3]+, a mimic of the Lys side chain and N-terminus, with common gas-phase modifiers (H2O, MeOH, EtOH, iPrOH, acetone, and MeCN) confirms that all solvent molecules form stable clusters at the site of protonation. Moreover, modeling populations of microsolvated clusters indicates that species containing protonated amine moieties exist as microsolvated species with one to six solvent ligands at all effective ion temperatures (Teff) accessible during a DMS experiment (ca. 375-600 K). Calculated Teff of protonated GGG, AAA, and Polybia-MPI using a modified two-temperature theory approach were up to 86 K cooler in DMS environments seeded with solvent vapor compared to pure N2 environments. Stabilizing effects were largely driven by an increase in the ion's apparent collision cross section and by evaporative cooling processes induced by the dynamic evaporation/condensation cycles incurred in the presence of an oscillating electric separation field. When the microsolvating partner was a protic solvent, abstraction of a proton from [MP1 + 3H]3+ to yield [MP1 + 2H]2+ was observed. This result was attributed to the proclivity of protic solvents to form hydrogen-bond networks with enhanced gas-phase basicity. Collectively, microsolvation provides analytes with a solvent "air bag," whereby charge reduction and microsolvation-induced stabilization were shown to shelter peptides from the fragmentation induced by field heating and may play a role in preserving native-like ion configurations.

Structure of the thiostrepton resistance methyltransferase.S-adenosyl-L-methionine complex and its interaction with ribosomal RNA.

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr.rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

Semi-synthetic analogues of thiostrepton delimit the critical nature of tail region modifications in the control of protein biosynthesis and antibacterial activity.

We report the successful production of selectively-modified tail analogues of the natural product antibiotic thiostrepton, which have been used to evaluate the critical nature of this section of the antibiotic to its inhibition of protein synthesis. This work highlights the tail region as a critical area for future semi-synthetic or synthetically bioengineered thiostrepton derivatives.

Design, synthesis and photocytotoxicity of upconversion nanoparticles: Potential applications for near-infrared photodynamic and photothermal therapy.

Photodynamic therapy (PDT) and photothermal therapy (PTT) are emerging modalities for the treatment of tumors and nonmalignant conditions, based on the use of photosensitizers to generate singlet oxygen or heat, respectively, upon light (laser) irradiation. They have potential advantages over conventional treatments, being minimally invasive with precise spatial-temporal selectivity and reduced side effects. However, most clinically employed PDT agents are activated at visible (vis) wavelengths for which the tissue penetration and, hence, effective treatment depth are compromised. In addition, the lipophilicity of near-infrared (NIR) photothermal agents limits their use and efficiency. To achieve combined PDT/PTT effects, both excitation wavelengths need to be tuned into the NIR spectral window of biological tissues. This paper reports the synthesis of neodymium-doped upconversion nanoparticles (NaYF4 :Yb,Er,Nd@NaYF4 :Nd) that convert 800 nm light into vis wavelengths, which can then activate conventional photosensitizers on the nanoparticle surface for PDT. Covalently bonded IR-780 dyes can readily be activated by 800 nm laser irradiation. The PEGylated nanoplatform exhibited a narrow size distribution, good stability and efficient generation of singlet oxygen under laser irradiation. The in vitro photocytotoxicity of this engineered nanoplatform as either a PDT or PTT agent in HeLa cells is demonstrated, while fluorescence microscopy in nanoplatform-incubated cells highlights its potential for bioimaging.

15N-1H HSQC NMR evidence for distinct specificity of two active sites in Escherichia coli glyoxalase I.

Much remains to be elucidated concerning the selectivity mechanism of supposedly identical active sites in oligomeric proteins. Glyoxalase I (GlxI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. The E. coli GlxI is a Ni(2+)/Co(2+)-activated homodimeric protein containing two symmetric, and dually metallated active sites as characterized by X-ray structure determination. Nevertheless, kinetics and isothermal titration calorimetric (ITC) studies indicate that dimeric GlxI binds to metal ions in a ratio of 1:1 (one metal ion/one dimer) [ Clugston , S. L. , Yajima , R. , and Honek , J. F. ( 2004 ) Biochem. J. 377 , 309 - 316 ]. In the current study, we provide spectroscopic evidence for the nonequivalent metallation of GlxI by use of (15)N-(1)H HSQC NMR titration experiments. (15)N-(1)H HSQC NMR spectra reveal that the local conformations of the two active sites in homodimeric GlxI are initially asymmetric in the apo-form, resulting in functional differentiation, wherein only one active site binds to the Ni(2+) ion, and another active site is observed to be more selective for a potent inhibitor. The current results enhance our understanding of GlxI structure-function relationships and provide a potential new strategy for the development of small molecule inhibitors for this enzyme system.

Pseudomonas aeruginosa contains multiple glyoxalase I-encoding genes from both metal activation classes.

The glyoxalase (Glx) system is a critical detoxification enzyme system that is widely distributed in prokaryotic and eukaryotic organisms. Glyoxalase I (GlxI), the first enzyme in the system, is a divalent metal-ion dependent lyase (isomerizing), and its homologs have recently been categorized into two metal activation classes which are either Zn2+-dependent or non-Zn2+ dependent (Ni2+-/Co2+-activated). The latter class encompasses enzymes of predominantly bacterial origin. We have identified two genes in Pseudomonas aeruginosa PAO1 encoding glyoxalase I enzymes in addition to the gloA1 sequence recently reported and characterized. The gloA1 and gloA2 genes encode non-Zn2+ dependent glyoxalase I enzymes and the gloA3 gene remarkably encodes a Zn2+-dependent homolog. To our knowledge this is the first report of a eubacterial species with several GlxI encoding genes, and also of an organism possessing GlxI enzymes from both metal activation classes.

Microbial glyoxalase enzymes: metalloenzymes controlling cellular levels of methylglyoxal.

The glyoxalase system consists of two enzymes, glyoxalase I and glyoxalase II. This system is important in the detoxification of methylglyoxal. Detailed studies have determined that the glyoxalase I from Escherichia coli, Neisseria meningitidis and Yersinia pestis are maximally activated by Ni2+ and Co2+, and are inactive with Zn2+, a situation quite different from the human glyoxalase I enzyme, which is activated by Zn2+. Recent studies on the Pseudomonas aeruginosa genome have led to the characterization of three different glyoxalase I enzymes, two of which follow a Ni2+/Co2+ activation profile and the third exhibits a human-like preference for Zn2+.

Emerging bacterial enzyme targets.

The treatment of bacterial infections is increasingly complicated by the ability of bacteria to develop resistance to antimicrobial agents, as well as by the emergence of new pathogens with the potential for rapid global spread. Thus, there is a critical need for novel antibacterial agents and new strategies to advance the drug discovery process. In the post-genomic era, comparative genomics, functional genomics and proteomics will play important roles in identifying new enzyme targets for the discovery of novel antibacterial agents. This review will discuss bacterial enzyme targets, specifically focusing on enzymes involved in fatty acid and cell wall biosynthesis.