Monomeric Enzyme Research Articles

Alanine Methyl Groups as NMR Probes of Molecular Structure and Dynamics in High-Molecular-Weight Proteins

The importance and utility of Ala(β) methyl groups as NMR probes of molecular structure and dynamics in high-molecular-weight proteins is explored. Using (2)H and (13)C relaxation measurements in {U-(2)H; Ala(β)-[(13)CHD(2)]}-labeled Malate Synthase G (MSG)--an 82-kDa monomeric enzyme that contains 73 Ala(β) methyl groups--we show that the vast majority of selectively labeled Ala(β) methyls are highly ordered. A number of NMR applications used for solution studies of structure and dynamics of large protein molecules can benefit from proximity of Ala(β) methyls to the protein backbone and their high degree of ordering. In the case of MSG, these applications include the measurement of (1)H-(13)C residual dipolar couplings in Ala(β) methyls, characterization of slow (μs-to-ms) dynamics at the substrates' binding sites, and methyl-TROSY-based NOE spectroscopy performed on {U-(2)H; Ala(β)-[(13)CH(3)]; Ile(δ1)-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples where the number of methyl probes for derivation of distance restraints is maximized compared to the state-of-the-art ILV labeling methodology.

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis: A MOLECULAR BASIS FOR UNDERSTANDING THE CONTROL OF YEAST HEXOKINASE FUNCTIONS VIA COVALENT MODIFICATION AND OLIGOMERIZATION

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ring-shaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. Two complex structures of KlHxk1 with bound glucose provide a molecular model of substrate binding to the open conformation and the subsequent classical domain closure motion of yeast hexokinases. The entirety of the novel data extends the current concept of glucose signaling in yeast and complements the induced-fit model by integrating the events of N-terminal phosphorylation and dissociation of homodimeric yeast hexokinases.

A Comparison of Thiol Peroxidase Mechanisms

Thiol peroxidases comprise glutathione peroxidases (GPx) and peroxiredoxins (Prx). The enzymes of both families reduce hydroperoxides with thiols by enzyme-substitution mechanisms. H(2)O(2) and organic hydroperoxides are reduced by all thiol peroxidases, most efficiently by SecGPxs, whereas fast peroxynitrite reduction is more common in Prxs. Reduction of lipid hydroperoxides is the domain of monomeric GPx4-type enzymes and of some Prxs. The catalysis starts with oxidation of an active-site selenocysteine (U(P)) or cysteine (C(P)). Activation of Cys (Sec) for hydroperoxide reduction in the GPx family is achieved by a typical tetrad composed of Cys (Sec), Asn, Gln, and Trp, whereas a triad of Cys Thr (or Ser) and Arg is the signature of Prx. In many of the CysGPxs and Prxs, a second Cys (C(R)) is required. In these 2-CysGPxs and 2-CysPrxs, the C(P) oxidized to a sulfenic acid forms an intra- or intermolecular disulfide (typical 2-CysPrx) with C(R), before a stepwise regeneration of ground-state enzyme by redoxin-type proteins can proceed. In SecGPxs and sporadically in Prxs, GSH is used as the reductant. Diversity combined with structural variability predestines thiol peroxidases for redox regulation via ROOH sensing and direct or indirect transduction of oxidant signals to specific protein targets.

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356-12358). In the ATP synthase dimer, the two peripheral stalks are located near the F(1)-F(1) interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.

Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V(max)) were 140 nanokatals mg(-1) for the linalool dehydratase and 410 nanokatals mg(-1) for the geraniol isomerase, with apparent K(m) values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).

High Resolution Measurement of Methyl 13Cm−13C and 1Hm−13Cm Residual Dipolar Couplings in Large Proteins

NMR methodology is developed for high-resolution, accurate measurements of methyl (1)H(m)-(13)C(m) ((1)D(CH)) and (13)C(m)-(13)C ((1)D(CC)) residual dipolar couplings (RDCs) in ILV-methyl-protonated high-molecular-weight proteins. Both types of RDCs are measured in a three-dimensional (3D) mode that allows dispersion of correlations to the third ((13)C(β/γ)) dimension, alleviating the problem of overlap of methyl resonances in highly complex and methyl-abundant protein structures. The methodology is applied to selectively ILV-protonated 82-kDa monomeric enzyme malate synthase G (MSG) that contains 273 ILV methyl groups with substantial overlap of methyl resonances in 2D methyl (1)H-(13)C correlation maps. A good agreement is observed between the measured RDCs of both types and those calculated from the crystallographic coordinates of MSG for the residues with low-amplitude internal dynamics. Although the measurement of (1)D(CH) RDCs from the acquisition dimension of NMR spectra imposes certain limitations on the accuracy of obtained (1)D(CH) values, (1)D(CH) couplings can be approximately corrected for cross-correlated relaxation effects. The ratios of (1)D(CH) and (1)D(CC) couplings ((1)D(CH)/(1)D(CC)) are independent of methyl axis dynamics and the details of residual alignment [Ottiger, M.; Bax, A. J. Am. Chem. Soc. 1999, 121, 4690.]. The (1)D(CH)/(1)D(CC) ratios obtained in MSG can therefore validate the employed correction scheme.

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity, a feature that facilitates enzyme-mediated regulation of blood glucose levels in the body. Two theoretical models have been proposed to describe the non-Michaelis-Menten behavior of human glucokinase. The mnemonic mechanism postulates the existence of one thermodynamically favored enzyme conformation in the absence of glucose, whereas the ligand-induced slow transition model (LIST) requires a preexisting equilibrium between two enzyme species that interconvert with a rate constant slower than turnover. To investigate whether either of these mechanisms is sufficient to describe glucokinase cooperativity, a transient-state kinetic analysis of glucose binding to the enzyme was undertaken. A complex, time-dependent change in enzyme intrinsic fluorescence was observed upon exposure to glucose, which is best described by an analytical solution comprised of the sum of four exponential terms. Transient-state glucose binding experiments conducted in the presence of increasing glycerol concentrations demonstrate that three of the observed rate constants decrease with increasing viscosity. Global fit analyses of experimental glucose binding curves are consistent with a kinetic model that is an extension of the LIST mechanism with a total of four glucose-bound binary complexes. The kinetic model presented herein suggests that glucokinase samples multiple conformations in the absence of ligand and that this conformational heterogeneity persists even after the enzyme associates with glucose.

Functional characterization of the PP2C phosphatase CaPtc2p in the human fungal pathogen Candida albicans

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ion. There are seven PP2C-like genes in Candida albicans. In this study, we demonstrate that CaPtc2p is a PP2C phosphatase. Surprisingly, in addition to the cytoplasmic localization, CaPtc2p is partially associated with mitochondria in yeast-form and filamentous cells of C. albicans. Expression of CaPTC2 is developmentally regulated during the serum-induced filamentation. Deletion of CaPTC2 renders C. albicans cells sensitive to SDS and azole antifungals, as well as the DNA methylation agent methylmethane sulphonate and the DNA synthesis inhibitor hydroxyurea. Therefore, CaPtc2p might fulfil multiple functions, including the regulation of mitochondrial physiology and checkpoint recovery from DNA damage in C. albicans cells.

Direct Evidence of Conformational Heterogeneity in Human Pancreatic Glucokinase from High-Resolution Nuclear Magnetic Resonance

High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.

Effects of ultraviolet radiation on the abundance, diversity and activity of bacterioneuston and bacterioplankton: insights from microcosm studies

The effects of ultraviolet-B (0.4 W m−2) radiation on the abundance, diversity and heterotrophic metabolism of bacterioneuston and bacterioplankton communities from Ria de Aveiro (Portugal) were assessed and compared to those of freshwater communities from Lake Vela (Portugal) in microcosm experiments. Exposure to 9 h of artificial ultraviolet radiation (UVR) led to 24–33% reduction in bacterial abundance and up to a 70% decrease in bacterial diversity. Maximum extracellular enzyme activity and monomer incorporation rates were reduced by 16–90% and 80–100%, respectively. Recovery of bacterial activity during post-UV dark incubations ranged from 10 to 100% for extracellular enzyme activity and 40% for monomer incorporation rates. In general, the heterotrophic activity of bacterioneuston was more inhibited by UVR than that of bacterioplankton. However, DGGE profiles revealed greater UVR-induced reductions in the diversity of bacterioplankton compared to bacterioneuston. The similarity between bacterioneuston and bacterioplankton communities in samples collected at early morning was lower than at noon (pre-exposed communities) and increased upon experimental irradiation, possibly indicating selection for UV-resistant bacteria. The observation that UV exposure resulted in enhanced reduction of bacterioneuston activity, but a lower reduction in bacterial diversity accompanied by enhanced dark recovery potential compared to bacterioplankton, indicates re-directioning of bacterioneuston metabolism towards stress defence/recovery strategies rather than the sustained heterotrophic metabolism. Our results indicate that UVR can significantly decrease the abundance, diversity and activity of bacteria inhabiting the surface and sub-surface layers of freshwater and estuarine systems with potentially important impacts on the biogeochemical cycles in these environments.

Production, purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l−1 in an optimized medium containing 20 g of malt extract l−1, 2 g of yeast extract l−1, 1.5 mM CuSO4. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (Km) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg2+, Mn2+, Zn2+ and Ca2+, while inhibited by 4.0 mM of Co2+, Al3+, Cu2+, and Fe2+, showing different profiles of metal ion effects.

Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

BackgroundTo examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC β1 and α subunits.Methodology/Principal FindingsCyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged α subunits with the carboxy-terminally tagged β1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the α subunits is close to the carboxy-terminus of the β1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO.Conclusions/SignificanceBased on the ability of an amino-terminal fragment of the β1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (αcatβcat), Winger and Marletta (Biochemistry 2005, 44:4083–90) have proposed a direct interaction of the amino-terminal region of β1 with the catalytic domains. In support of such a concept of “trans” regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed “fluorescent-conjoined” sGC's by fusion of the α amino-terminus to the β1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.

Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 β-lactamase

In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) beta-lactamase. The first one, cpTEM-1-His(3) was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn(2+). The second one, cpTEM-1-3M-His(2), was selected by a directed evolution strategy. It is allosterically down-regulated by Zn(2+), Ni(2+) and Co(2+) with binding affinities around 300 microM.

Crystallization and preliminary crystallographic analysis of Gre2p, an NADP(+)-dependent alcohol dehydrogenase from Saccharomyces cerevisiae.

Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP(+). Crystals of a Gre2p complex with NADP(+) were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P2(1). The current diffraction resolution is 3.2 A. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis.

Molecular Architecture of a C-3′-Methyltransferase Involved in the Biosynthesis of d-Tetronitrose

S-Adenosylmethionine (SAM)-dependent methyltransferases are involved in a myriad of biological processes, including signal transduction, chromatin repair, metabolism, and biosyntheses, among others. Here we report the high-resolution structure of a novel C-3'-methyltransferase involved in the production of D-tetronitrose, an unusual sugar found attached to the antitumor agent tetrocarcin A or the antibiotic kijanimicin. Specifically, this enzyme, referred to as TcaB9 and cloned from Micromonospora chalcea, catalyzes the conversion of dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose to dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-D-glucose. For this analysis, two structures were determined to 1.5 A resolution: one in which the enzyme was crystallized in the presence of SAM and dTMP and the other with the protein complexed to S-adenosylhomocysteine and its dTDP-linked sugar product. The overall fold of the monomeric enzyme can be described in terms of three domains. The N-terminal domain harbors the binding site for a zinc ion that is ligated by four cysteines. The middle domain adopts the canonical "SAM-binding" fold with a seven-stranded mixed beta-sheet flanked on either side by three alpha-helices. This domain is responsible for anchoring the SAM cofactor to the protein. Strikingly, the C-terminal domain also contains a seven-stranded beta-sheet, and it appears to be related to the middle domain by an approximate 2-fold rotational axis, thus suggesting TcaB9 arose via gene duplication. Key residues involved in sugar binding include His 181, Glu 224, His 225, and Tyr 222. Their possible roles in catalysis are discussed.

Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a approximately 21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

Binding Selectivity of RecA to a single stranded DNA, a computational approach

Homologous recombination (HR) is the major DNA double strand break repair pathway which maintains the genomic integrity. It is fundamental for the survivability and functionality of all organisms. One of the initial steps in HR is the formation of the nucleoprotein filament composed by a single stranded DNA chain surrounded by the recombinases protein. The filament orchestrates the search for an undamaged homologue, as a template for the repair process. Our theoretical study was aimed at elucidating the selectivity of the interaction between a monomer of the recombinases enzyme in the Escherichia coli, EcRecA, the bacterial homologue of human Rad51, with a series of oligonucleotides of nine bases length. The complex, equilibrated for 20 ns with Langevian dynamics, was inserted in a periodic box with a 8 Å buffer of water molecules explicitly described by the TIP3P model. The absolute binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann (PB) and the generalized Born (GB) solvent accessible surface area, using the MM-PB(GB)SA model. The solute entropic contribution is also calculated by normal mode analysis. The results underline how a significant contribution of the binding free energy is due to the interaction with the Arg196, a critical amino acid for the activity of the enzyme. The study revealed how the binding affinity of EcRecA is significantly higher toward dT₉ rather than dA₉, as expected from the experimental results.

Gene cloning and characterization of a novel thermophilic esterase from <italic>Fervidobacterium nodosum</italic> Rt17-B1

A bioinformatic screening of the genome of the thermophilic bacterium Fervidobacterium nodosum Rt17-B1 for esterhydrolyzing enzymes revealed a putative bacterial esterase (FNE) encoded by Fond_1301 with typical GDSL family motifs. To confirm its putative esterase function, the FNE gene was cloned, functionally expressed in Escherichia coli, and purified to homogeneity. Recombinant FNE exhibited the highest esterase activity of 14,000 U/mg with p-nitrophenyl acetate (pNPC(2)) as substrate. The catalytic efficiency (k(cat)/K(m)) toward p-nitrophenyl acetate (C(2)) was approximately 120-fold higher than toward p-nitrophenyl butyrate (C(4)). No significant esterase activity was observed for the substrates with a chain length > or =C(8). The monomeric enzyme has a molecular mass of 27.5 kDa and exhibits optimal activity around 75 degrees C, at pH 8.5. Its thermostability is relatively high with a half-life of 80 min at 70 degrees C, but less stable compared with some other hyperthermophilic esterases. A structural model was constructed using acetylesterase from Aspergillus aculeatus as a template. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine, which was verified by site-directed mutagenesis. Sequence analysis showed that FNE was only distantly related to other esterases. A comparison of the conserved motifs shared with GDSL proteins revealed that FNE could be grouped into GDSL family and was further classified as SGNH hydrolase.

Hopping Enables a DNA Repair Glycosylase To Search Both Strands and Bypass a Bound Protein

Spontaneous DNA damage occurs throughout the genome, requiring that DNA repair enzymes search each nucleotide every cell cycle. This search is postulated to be more efficient if the enzyme can diffuse along the DNA, but our understanding of this process is incomplete. A key distinction between mechanisms of diffusion is whether the protein maintains continuous contact (sliding) or whether it undergoes microscopic dissociation (hopping). We describe a simple chemical assay to detect the ability of a DNA modifying enzyme to hop and have applied it to human alkyladenine DNA glycosylase (AAG), a monomeric enzyme that initiates repair of alkylated and deaminated purine bases. Our results indicate that AAG uses hopping to effectively search both strands of a DNA duplex in a single binding encounter. This raised the possibility that AAG might be capable of circumnavigating blocks such as tightly bound proteins. We tested this hypothesis by binding an EcoRI endonuclease dimer between two sites of DNA damage and measuring the ability of AAG to act at both damaged sites in a single binding encounter. Remarkably, AAG bypasses this roadblock in approximately 50% of the binding events. We infer that AAG makes significant excursions from the surface of the DNA, allowing reorientation between strands and the bypass of a bound protein. This has important biological implications for the search for DNA damage because eukaryotic DNA is replete with proteins and only transiently accessible.

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.