Does kidney biopsy in pediatric lupus patients "complement" the management and outcomes of silent lupus nephritis? Lessons learned from a pediatric cohort.

BACKGROUND: Silent lupus nephritis (SLN) is systemic lupus erythematosus (SLE) without clinical and laboratory features of kidney involvement but with biopsy-proven nephritis. This study aims to describe and compare the baseline characteristics and outcomes of pediatric SLN with overt LN (OLN) and to identify associated risk factors and biochemical markers. METHODS: In this retrospective, observational study, multivariate logistic regression and receiver operating characteristic (ROC) analyses studied age, sex, race, serum complements, anti-double-stranded-DNA antibody, anti-Smith antibody, eGFR, and proliferative nephritis. RESULTS: In our cohort of 69 patients, 47 were OLN, and 22 were SLN. OLN (OR = 4.9, p = 0.03) and non-African Americans (AA) (OR = 13.0, p < 0.01) had higher odds, and increasing C3 and C4 were associated with lower odds of proliferative nephritis (OR 0.95 and 0.65 per one unit increase in C3 and C4, respectively, p < 0.01). They demonstrated a good discriminative ability to detect proliferative nephritis as assessed by the area under the ROC curve (C3 = 0.78, C4 = 0.78). C3 and C4 in proliferative SLN and OLN were comparable and significantly lower than their non-proliferative counterparts. No association was observed between age, sex, anti-double-stranded-DNA antibody, anti-Smith antibody, eGFR, and proliferative nephritis. Proliferative SLN and OLN patients received similar treatments. Adverse events were identified in the proliferative OLN only. CONCLUSIONS: Lower complement levels are associated with proliferative lesions in pediatric LN-both SLN and OLN. The non-AA population had higher odds of having proliferative nephritis than the AA. Prospective, randomized, long-term follow-up of proliferative SLN patients is needed to ascertain the beneficial effect of early diagnosis and treatment. A higher resolution version of the Graphical abstract is available as Supplementary information.

High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbß Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor.

Rev-erbα and Rev-erbß are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbß (FLRev-erbß) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbß·DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbß·NCoR1 complex. The interaction between FLRev-erbß and NCoR1 as well as Rev-erbß repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erbß in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10(-6) s(-1)) of heme dissociation from Rev-erbß rules out a prior proposal that Rev-erbß acts as an intracellular heme sensor.

Structural asymmetry of the terminal catalytic complex in selenocysteine synthesis.

Selenocysteine (Sec), the 21(st) amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNA(Sec)). In archaea and eukaryotes, O-phosphoseryl-tRNA(Sec):selenocysteinyl-tRNA(Sec) synthase (SepSecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while being attached to tRNA(Sec). We have previously shown that only the SepSecS tetramer is capable of binding to and recognizing the distinct fold of tRNA(Sec). Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised regarding whether the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small angle x-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between SepSecS and tRNA(Sec) in vivo. We show that SepSecS preferentially binds one or two tRNA(Sec) molecules at a time and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between SepSecS and two tRNAs, one enzyme homodimer plays a role of the noncatalytic unit that positions CCA ends of two tRNA(Sec) molecules into the active site grooves of the other, catalytic, homodimer. Finally, our results demonstrate that the previously determined crystal structure represents the physiologically and catalytically relevant complex and suggest that allosteric regulation of SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.

The RIVUR study: a review of its findings.

BACKGROUND: The conclusion drawn by the authors of the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) trial is that antimicrobial prophylaxis reduces the risk of recurrent urinary tract infection (UTI)-but not of renal scarring-in patients with vesicoureteral reflux (VUR). RESULTS: A review of the findings showed that the decreased recurrent UTI rate was only present at the end of the 2-year follow-up period and was only slightly increased (12.3%) above the 10% cutoff for statistical significance. The difference was not observed in children younger than two years of age with VUR grade III and IV. In addition, the rate of new renal scarring was not statistically different between the prophylaxis and placebo groups (8.2 vs. 8.4%, respectively). A high rate of uropathogen antibiotic resistance was observed in the prophylaxis group (68.4 vs. 24.6%, respectively). CONCLUSION: The analysis of the RIVUR findings questions the validity of its authors suggestion that the results may warrant reconsideration of the current recommendations by the American Academy of Pediatrics on obtaining a voiding cystourethrogram after the first febrile UTI and the use of urinary antibiotic prophylaxis in VUR patients.

The selenocysteine-specific elongation factor contains a novel and multi-functional domain.

The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA(Sec)) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA(Sec) binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA(Sec) binding and accommodation into the ribosomal A-site.

Thiol-disulfide redox dependence of heme binding and heme ligand switching in nuclear hormone receptor rev-erb{beta}.

Rev-erbß is a heme-binding nuclear hormone receptor that represses a broad spectrum of target genes involved in regulating metabolism, the circadian cycle, and proinflammatory responses. Here, we demonstrate that a thiol-disulfide redox switch controls the interaction between heme and the ligand-binding domain of Rev-erbß. The reduced dithiol state of Rev-erbß binds heme 5-fold more tightly than the oxidized disulfide state. By means of site-directed mutagenesis and by UV-visible and EPR spectroscopy, we also show that the ferric heme of reduced (dithiol) Rev-erbß can undergo a redox-triggered switch from imidazole/thiol ligation (via His-568 and Cys-384, based on a prior crystal structure) to His/neutral residue ligation upon oxidation to the disulfide form. On the other hand, we find that change in the redox state of iron has no effect on heme binding to the ligand-binding domain of the protein. The low dissociation constant for the complex between Fe(3+)- or Fe(2+)-heme and the reduced dithiol state of the protein (K(d) = ∼ 20 nM) is in the range of the intracellular free heme concentration. We also determined that the Fe(2+)-heme bound to the ligand-binding domain of Rev-erbß has high affinity for CO (K(d) = 60 nM), which replaces one of the internal ligands when bound. We suggest that this thiol-disulfide redox switch is one mechanism by which oxidative stress is linked to circadian and/or metabolic imbalance. Heme dissociation from Rev-erbß has been shown to derepress the expression of target genes in response to changes in intracellular redox conditions. We propose that oxidative stress leads to oxidation of cysteine(s), thus releasing heme from Rev-erbß and altering its transcriptional activity.

Redox, haem and CO in enzymatic catalysis and regulation.

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 Å (1 Å=0.1 nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.

Minimal Change Disease as Initial Presentation of ALK-Positive Anaplastic Large-Cell Lymphoma in a Pediatric Patient.

The association between nephrotic syndrome (NS), hemophagocytic lymphohistiocytosis (HLH), and certain paraneoplastic syndromes has been documented in the literature. However, nephrotic changes as part of paraneoplastic syndromes are rare in lymphoid malignancies, particularly in non-Hodgkin lymphoma. We report the sudden onset of acute renal failure and NS in a 14-year-old male who initially presented with HLH and was subsequently diagnosed with ALK-positive anaplastic large-cell lymphoma (ALCL). The treatment of ALCL reversed both the HLH and NS findings. This case demonstrates the importance of considering lymphoma in pediatric patients presenting with NS and HLH.

Use of C4d as a diagnostic tool to classify membranoproliferative glomerulonephritis.

BACKGROUND: Membranoproliferative glomerulonephritis (MPGN type I, II and III) was reclassified in 2013 as MPGN and C3 glomerulopathy (C3G) based on the complement system activation mechanism. OBJECTIVES: To evaluate whether C4d, a component of the classical pathway, could be a diagnostic tool in differentiating between MPGN and C3G. METHODS: We conducted a retrospective study of 15 MPGN type I, II and III and 13 minimal change disease (MCD) patients diagnosed between 2000 and 2012. C4d staining using the peroxidase method was employed. RESULTS: Using the 2013 C3G consensus classification, the 15 MPGN types I, II and III biopsies were re-classified as MPGN (8) and C3G (7). Following C4d staining, of the 8 biopsies diagnosed as MPGN, 4 had classical pathway involvement [C1q (+), C3 (+), C4d (+)]; two had lectin pathway involvement [C1q (-), C3 (+), C4d (+)]; and, two were reclassified as C3G because the absence of C4d and C1q suggested the presence of the alternative pathway [C1q (-), C3 (+), C4d (-)]. Three of the seven C3G biopsies presented classical pathway involvement and were reclassified as MPGN. The alternative pathway was present in one of the other 4 biopsies considered to be C3G. Two C3G biopsies involved the lectin pathway and the one case of dense deposit disease had lectin pathway involvement. CONCLUSIONS: C4d staining may help to differentiate between MPGN and C3G. In addition, the lectin pathway could play a role in the pathogenesis of these glomerulopathies.

The utilization of selenocysteine-tRNA[Ser]Sec isoforms is regulated in part at the level of translation in vitro.

The tRNA for the 21st proteinogenic amino acid, selenocysteine, exists in mammalian cells as 2 isoforms differing by a single 2'-O-methylribosyl moiety at position 34 (Um34). These isoforms contain either 5-methoxycarbonylmethyluridine (mcm5U) or 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) at position 34. The accumulation of the mcm5Um isoform is tightly correlated with the expression of nonessential "stress response" selenoproteins such as glutathione peroxidase 1 (GPX1). The expression of essential selenoproteins, such as thioredoxin reductase 1 (TXNRD1), is not affected by changes in Sec-tRNA[Ser]Sec isoform accumulation. In this work we used purified mcm5U and mcm5Um Sec-tRNA[Ser]Sec isoforms to analyze possible differences in binding to the selenocysteine-specific elongation factor, EEFSEC, and the translation of GPX1 and TXNRD1in vitro. Our results indicate that no major distinction between mcm5U and mcm5Um isoforms is made by the translation machinery, but a small consistent increase in GPX1 translation is associated with the mcm5Um isoform. These results implicate fundamental differences in translation efficiency in playing a role in regulating selenoprotein expression as a function of isoform accumulation.

BK viruria and viremia in children with systemic lupus erythematosus.

BACKGROUND: BK virus (BKV) is a ubiquitous polyoma virus that lies dormant in the genitourinary tract once acquired in early childhood. In states of cellular immunodeficiency, the virus can reactivate to cause hemorrhagic cystitis and nephritis. Children with systemic lupus erythematosus (SLE) have an increased risk of developing infectious complications secondary to their immunocompromised state from the administration of several immuno-modulatory drugs. Currently, there are no data regarding the prevalence of BK viruria or viremia in children with SLE. METHODS: We conducted a prospective cohort study involving children with SLE of 18 years and younger. We obtained urine and blood samples at baseline and every 3 months up to 1 year for BK virus detection by real-time, quantitative polymerase chain reaction analysis. A comprehensive review of demographic information, clinical characteristics and medication history was also obtained. RESULTS: Thirty-two pediatric patients (26 females and 6 males) with SLE were enrolled. Median age at the time of SLE diagnosis and enrollment into study was 13.6 years and 16.0 years old, respectively. The prevalence at enrollment was 3.1% (1/32) for BK viruria and 6.2% (2/32) for BK viremia. During the study period, 3 patients had viruria, 5 had viremia and 4 had both viruria and viremia. Of the 12 patients with BKV reactivation, only one was positive for microscopic hematuria, all others were asymptomatic. A total of nine of 97(9.2%) urine samples and 10 of 96(10.4%) blood samples were positive for BK virus. The most commonly utilized biologics in this cohort group were Rituximab (90.6%), Abatacept (12.5%), and Belimumab (9.3%). The type of medication exposure and clinical characteristics did not statistically differ between the groups that did or did not have BK viruria and/or viremia. CONCLUSIONS: Our study suggests that pediatric patients with SLE have BK viremia and/or viruria at a higher rate than the general healthy population, although the significance of the reactivation and viral level is unclear. The influence of immune-modulatory drugs on BKV reactivation is still uncertain. To understand the interplay amongst BK virus, immunosuppression and dysregulated immune system in children with SLE, ongoing research in a larger population is still warranted, which may help establish proper surveillance, diagnosis and treatment for BKV infection.

Single-cell antibody nanowells: a novel technology in detecting anti-SSA/Ro60- and anti-SSB/La autoantibody-producing cells in peripheral blood of rheumatic disease patients.

BACKGROUND: Anti-SSA/Ro60 and anti-SSB/La are essential serological biomarkers for rheumatic diseases, specifically Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Currently, laboratory detection technology and platforms are designed with an emphasis on high-throughput methodology; therefore, the relationship of sensitivity with specificity remains a significant area for improvement. In this study, we used single-cell antibody nanowells (SCAN) technology to directly profile individual B cells producing antibodies against specific autoantigens such as SSA/Ro60 and SSB/La. METHODS: Peripheral blood mononuclear cells were isolated using Ficoll gradient. Fluorescently labeled cells were added to fabricated nanowells and imaged using a high-speed epifluorescence microscope. The microengraving process was conducted using printed slides coated with immunoglobulins. Printed slides were hybridized with fluorescence-conjugated immunoglobulin G (IgG), SSA/Ro60, and SSB/La antigens. Microarray spots were analyzed for nanowells with single live B cells that produced antigen-specific autoantibodies. RESULTS: Our results indicate that SCAN can simultaneously detect high frequencies of anti-SSA/Ro60 and anti-SSB/La with a specific IgG isotype in peripheral blood mononuclear cells of patients, as well as measure their individual secretion levels. The data showed that patients with SS and SLE exhibited higher frequency and greater concentration of anti-SSA/Ro60- and anti-SSB/La-producing B cells in the IgG isotype. Furthermore, individual B cells of patients produced higher levels of IgG-specific anti-SSA/Ro60 autoantibody, but not IgG-specific anti-SSB/La autoantibody, compared with healthy control subjects. CONCLUSIONS: These results support the application of SCAN as a robust multiparametric analytical bioassay that can directly measure secretion of autoantibody and accurately report antigen-specific, autoantibody-producing cells.

Beta-catenin-mediated transactivation and cell-cell adhesion pathways are important in curcumin (diferuylmethane)-induced growth arrest and apoptosis in colon cancer cells.

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.

Review article: BK virus in systemic lupus erythematosus.

BK virus (BKV) is a human polyomavirus with a seroprevalence of 60-80 % in the general population. In renal transplant patients, it is known to cause renal failure, ureteric stenosis and hemorrhagic cystitis. In bone marrow transplant patients, it is evident that BKV can also cause hemorrhagic cystitis along with BK virus nephropathy (BKVN) in the native kidneys, with subsequent renal failure. However, little is known about BVKN in non-transplanted immune-compromised patients, such as systemic lupus erythematosus (SLE) who may have underlying nephritis and have a compromised immune system due to therapy and/or systemic illness. Thus, this article will focus on the clinical aspects of BKV and its association in patients with SLE.

Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements.

Selenoproteins are present in all three domains of life and are responsible for a major part of a cell's antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: the Sec insertion sequence (SECIS) element in the 3' untranslated region, Sec-tRNA(Sec), the Sec-specific elongation factor eEFSec, and SECIS binding protein 2. Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here, we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNA(Sec) and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec-utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation.

Use of C4d as a diagnostic tool to classify membranoproliferative glomerulonephritis / Uso de C4d como herramienta diagnóstica para clasificar la glomerulonefritis membranoproliferativa

No disponible

Late Diagnosis of Fanconi-Bickel Syndrome: Challenges With the Diagnosis and Literature Review

Abstract Fanconi-Bickel syndrome (FBS), also known as glycogen storage disease type XI (GSD XI), is a rare autosomal recessive disorder of carbohydrate metabolism. It is caused by mutations in the gene SLC2A2, which encodes for the facilitative glucose transporter GLUT2. Diagnosis of FBS is often delayed since the clinical features and laboratory markers often overlap with other disorders whose characteristic features include short stature, fasting hypoglycemia, postprandial hyperglycemia, hepatomegaly, hypophosphatemic rickets, and proximal renal tubular dysfunction. In this article, we present a case of FBS and its management in an African American female who initially presented with persistent proximal tubulopathy, hypercalciuria, and metabolic acidosis. We also include a recent literature review on FBS and discuss other metabolic disorders that should be considered in the differential diagnosis.

Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores.

Directed evolution is an effective strategy to engineer and optimize protein properties, and microbial cell-surface display is a successful method to screen protein libraries. Protein surface display on Bacillus subtilis spores is demonstrated as a tool for screening protein libraries for the first time. Spore display offers advantages over more commonly utilized microbe cell-surface display systems, which include gram-negative bacteria, phage and yeast. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; hence, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. The substrate specificity of the enzyme CotA is narrowed. CotA is a laccase and it occurs naturally on the outer coat of B. subtilis spores. A library of CotA genes were expressed in the spore coat, and it was screened for activity toward ABTS [diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] over SGZ (4-hydroxy-3,5-dimethoxy-benzaldehyde azine). A mutant CotA was found to be 120-fold more specific for ABTS. This research demonstrates that B. subtilis spores can be a useful platform for screen protein libraries.

Narrowing laccase substrate specificity using active site saturation mutagenesis.

The laccase CotA from Bacillus subtilis was converted from a generalist, an enzyme with broad specificity, to a specialist, an enzyme with narrowed specificity. Laccases are members of the multicopper oxidase family and have many applications in biotechnology. To date, it has not been demonstrated that substrate specificity can be tapered for a laccase. Wild-type CotA oxidizes ABTS (ABTS = diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ = 4-hydroxy-3,5- dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS by combining rational and directed evolution approaches. The wild-type was evolved by simultaneously randomizing 19 amino acids found in the substrate-binding pocket. A mutant was identified that had a catalytic efficiency, (k(cat)/K(M))(ATBS) / (k(cat)/K(M))(SGZ), 7.0 times greater when compared to the wild-type after one round of screening. This illustrates that the substrate-binding pocket is highly evolvable for specificity.

Dual roles of an essential cysteine residue in activity of a redox-regulated bacterial transcriptional activator.

CprK from Desulfitobacterium dehalogenans is the first characterized transcriptional regulator of anaerobic dehalorespiration and is controlled at two levels: redox and effector binding. In the reduced state and in the presence of chlorinated aromatic compounds, CprK positively regulates expression of the cpr gene cluster. One of the products of the cpr gene cluster is CprA, which catalyzes the reductive dehalogenation of chlorinated aromatic compounds. Redox regulation of CprK occurs through a thiol/disulfide redox switch, which includes two classes of cysteine residues. Under oxidizing conditions, Cys11 and Cys200 form an intermolecular disulfide bond, whereas Cys105 and Cys111 form an intramolecular disulfide. Here, we report that Cys11 is involved in redox inactivation in vivo. Upon replacement of Cys11 with serine, alanine, or aspartate, CprK loses its DNA binding activity. C11A is unstable; however, circular dichroism studies demonstrate that the stability and overall secondary structures of CprK and the C11S and C11D variants are similar. Furthermore, effector binding remains intact in the C11S and C11D variants. However, fluorescence spectroscopic results reveal that the tertiary structures of the C11S and C11D variants differ from that of the wild type protein. Thus, Cys11 plays a dual role as a redox switch and in maintaining the correct tertiary structure that promotes DNA binding.