Protein Modeling Studies Research Articles

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss.

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6. Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene.

Development of a Cucumis sativus TILLinG platform for forward and reverse genetics.

BackgroundCucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics.Principal FindingsA population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation.ConclusionsWe have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

Novel Decorin Mutation in a Chinese Family With Congenital Stromal Corneal Dystrophy

The aim of this study was to characterize the congenital stromal corneal dystrophy (CSCD) pathological and clinical phenotype in a Chinese family with a novel mutation of decorin and its possible molecular pathogenesis. Molecular genetic analyses were performed on 5 patients with CSCD. Clinical characteristics, optical coherence tomography, and confocal microscopic study were evaluated. The corneal specimens from patients with CSCD were sent for light and electron microscopic evaluation. A protein modeling study was carried out to assess the effect of the mutation on the protein structure. Sequencing analysis of DCN revealed that all patients with CSCD were heterozygous for a 1-bp deletion at nucleotide 962 (c.962delA) in exon 8. This causes a premature termination of the decorin protein by frameshift, causing the deletion of 33 amino acids in the C-terminal end of the decorin protein. Optical coherence tomography and confocal microscopic study demonstrated that the corneal lamellar structure was disrupted and that this is more severe in the anterior and posterior central stroma. Histopathological study showed that electron-lucent zones were present between the normal-appearing collagen lamellae in the patients with CSCD. Abnormally thinned collagen filaments were identified in the electron-lucent zones, which could be due to abnormal decorin binding to the collagen microfibrils. Protein modeling studies involving wild-type and mutant protein indicated that mutant decorin might be unable to bind to all 4 collagen microfibrils as the normal decorin would. We present the clinical, histopathological, and molecular genetic assessment of a Chinese family with CSCD in which a novel DCN mutation was identified. Our findings add to the allelic heterogeneity of this rare form of inherited corneal disease.

Evolved Quorum Sensing Regulator, LsrR, for Altered Switching Functions

In order to carry out innovative complex, multistep synthetic biology functions, members of a cell population often must communicate with one another to coordinate processes in a programmed manner. It therefore follows that native microbial communication systems are a conspicuous target for developing engineered populations and networks. Quorum sensing (QS) is a highly conserved mechanism of bacterial cell-cell communication and QS-based synthetic signal transduction pathways represent a new generation of biotechnology toolbox members. Specifically, the E. coli QS master regulator, LsrR, is uniquely positioned to actuate gene expression in response to a QS signal. In order to expand the use of LsrR in synthetic biology, two novel LsrR switches were generated through directed evolution: an "enhanced" repression and derepression eLsrR and a reversed repression/derepression function "activator" aLsrR. Protein modeling and docking studies are presented to gain insight into the QS signal binding to these two evolved proteins and their newly acquired functionality. We demonstrated the use of the aLsrR switch using a coculture system in which a QS signal, produced by one bacterial strain, is used to inhibit gene expression via aLsrR in a different strain. These first ever AI-2 controlled synthetic switches allow gene expression from the lsr promoter to be tuned simultaneously in two distinct cell populations. This work expands the tools available to create engineered microbial populations capable of carrying out complex functions necessary for the development of advanced synthetic products.

Homology Modeling, Molecular Docking and DNA Binding Studies of Nucleotide Excision Repair UvrC Protein from M. tuberculosis

Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.

Identification of functionally relevant lysine residues that modulate human farnesoid X receptor activation.

Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter α/β and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ∼21%. K460R decreased the FXR interaction with Set7/9 by ∼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ∼80 and ∼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).

Protein modeling and molecular dynamic studies of two new surfactant proteins

Surfactant proteins are of major importance for the stability and flexibility of lipid systems like the lung surfactant or the tear film. They can support the adsorption of phospholipids into a layer or specifically influence the surface tension of a lipid surface [1]. To fulfil these functions, they are described to be highly posttranslationally modified. Furthermore, immunological functions were described for some of the already known surfactant proteins SP-A, SP-B, SP-C and SP-D [2]. For that reason, they are of great interest in the investigation of diseases like the acute respiratory distress syndrome (ARDS) or the dry eye syndrome (DES). Recently sequences of two putative surfactant proteins, called SP-G and SP-H, were identified. To get insights into the function of SP-G and SP-H, protein structure models were generated, including predictions for posttranslational modifications. These models successfully guided the design of antibodies for the localization of SP-G and SP-H in different human tissues, including lung and ocular system. Both protein models were transferred into a lung surfactant model system consisting of a dipalmitoyl¬phosphatidyl¬choline monolayer. MD simulations over 50 ns were performed with GROMACS starting from different orientations of the protein models. During these simulations, it was possible to track the accumulation of the proteins to the lipid layer and observe the interactions between protein surface and lipid head groups on an atomic scale. The results show that SP-G and SP-H indeed are capable of surface-regulatory properties. Furthermore, it is evident that the interaction between proteins and lipid systems is highly dependent on the present posttranslational modifications. In combination with further experimental work, these simulations can help to determine the functions of SP-G and SP-H in vivo and may support the development of new therapies for ARDS and DES.

Molecular Modeling and Interaction Studies of HCV Core Protein

HCV is a leading cause of liver disease and can lead to hepatocellular carcinoma and liver damage which in turn can cause death of patients. HCV is closely related to immune response especially inflammation suggesting that the immune response-related genes can serve as molecular targets for chemo-prevention and treatment of C-type HCC. This study was conducted to determine the 3D structures of immune responsive gene (CXCL6) that are up-regulated during Hepatitis C Virus (HCV) Infection and Hepatocellular Carcinoma (HCC) and HCV core protein.Furthermore, docking and interactions analysis of immune responsive gene with HCV core protein was performed to understand pathogenesis of HCV infection. Reliable 3D structures of both proteins were determined using comparative modeling approach. Docking of both proteins revealed functionally important residues i-e. Arg149, Arg39, Arg74 and Gln78 in HCV core protein and Leu44, Ala71, Ser76 and Pro97 in CXCL6. It can be concluded from the study that CXCL6 had potentially interacting residues with HCV core protein that can be helpful in finding clues to understand HCV pathogenesis and develop better therapeutic regimens.

Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae.

Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of L-lysine and L-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants.

3-Dimentional molecular modeling studies of KSHV vOX2 protein as an example of highly glycosylated and adhesive molecule

Introduction: KSHV which is the etiological agent of Kaposi's Sarcoma encodes a protein by open reading frame (ORF) K14, called vOX2. This protein shares identity with the mammalian OX2 (CD200). Signal delivery of vOX2 occurs through binding to CD200R which contributes to maintaining the homeostasis of immune responses, in a manner similar to CTLA-4 and PDL-1/-2. Due to highly glycosylation and adhesive properties of vOX2, it failed to determine the 3D-structure of the protein by Xray crystallography method. Therefore, probable crystal structure of vOX2 has been bioinformatically identified. Material and methods: In order to identify the templates, BLAST sequence homology searches (NCBI) were performed. The PD-L1 (PDB entry: 3FN3) was chosen for modeling. Multiple alignment process was carried out on the selected sequences by ClustalX2 (protein weight matrix: BLOSUM series). Model building was performed in the program MODELLER9v5 using model-ligand algorithm and models at various refinement levels were generated. Finally the refined structures were minimized under molecular mechanic AMBER method (RMS gradient = 0.5) in HyperChem7.5. All models were validated using the program ERRAT and PROCHECK at UCLA. Results: The best 3D model had an Errat score of 94%. In the modelled protein all of the glycosyl-linked residues (Asn 83, 91, 138, 157 and 166) are situated in the external region of the 3D structure with steric flexibility of their side chains. Conclusion: Our modeling supports the data which indicates that vOX2 has two Ig-like domains, located between residue 9 to 120, and 130–160, respectively and the integrin-binding motif, RGD, at residues 190–193 is exposed.

WHERE IS THE DEIODINASE SELECTIVITY FOR ORD OR IRD LOCATED? A STRUCTURE-FUNCTION APPROACH

WHERE IS THE DEIODINASE SELECTIVITY FOR ORD OR IRD LOCATED? A STRUCTURE-FUNCTION APPROACH

Molecular and structural analysis of metachromatic leukodystrophy patients in Indian population

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by mutations in arylsulfatase A ( ARSA) gene. No work on molecular genetics of MLD has been reported from India and the mutational spectrum in Indian patients is not known. The present study was undertaken to identify mutations in arylsulfatase A gene in Indian MLD patients, to evaluate genotype–phenotype correlation, and to see the effect of the novel mutants on the protein. Twenty MLD patients (16 families) were screened by ARMS PCR for the most common mutation (c.459+1G>A). Pseudodeficiency alleles were tested by RFLP method whereas rare and novel mutations were scanned by Conformation Sensitive Gel Electrophoresis (CSGE), followed by sequencing. The genotype–phenotype correlation was also attempted. Protein homology modeling analysis was carried out for two novel missense mutations identified, to assess the effect of these mutations on the protein conformation. Nine pathogenic alleles were found in 13 patients (65%). Four previously reported mutations and five novel variants were identified. Five patients (35%) were found to have pseudodeficiency alleles, c.1049A>G (p.Asn350Ser) and c.1524+95A>G. Genotype–phenotype correlation was found to be difficult to establish. Protein modeling studies showed that the mutations cause loss of interactions leading to conformational change in ASA protein. The study identified the mutational spectrum of Indian MLD patients, which will be helpful in genetic counseling, carrier detection and establishing prenatal diagnosis. Homology modeling helped to study conformational change in protein and has implications in generating novel therapeutic molecules.

Homology Modeling and Docking Studies of Human Bcl-2L10 Protein

Cancer, an unrestrained proliferation of cells, is one of the lead cause of death. Nearly 12.5 million people are diagnosed with cancer worldwide, 7.5 million people die of which 2.5 million cases are from India. Major cause for cancer is restriction of programmed cell death (apoptosis). Multiple signaling pathways regulate apoptosis. Bcl-2 (B—Cell Lymphomas-2) family proteins play a vital role as central regulators of apoptosis. Bcl-2L10, a novel anti- apoptotic protein, blocks apoptosis by mitochondrial dependent mechanism. The present study evaluates the 3D structure of Bcl-2L10 protein using homology modeling and aims to understand plausible functional and binding interactions between Bcl-2L10 with BH3 domain of BAX using protein—protein docking. The docking studies show binding of BH3 domain at Lys 110, Trp-111, Pro-115, Glu-119 and Asp-127 in the groove of BH 1, 2 and 3 domains of Bcl-2L10. Heterodimerization of anti-apoptotic Bcl-2 and BH3 domain of pro- apoptotic Bcl-2 proteins instigates apoptosis. Profound understanding of Bcl-2 pathway may prove useful in identification of future therapeutic targets for cancer.

Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase

A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the alphaL helix-capping motif at the C-terminus of the alpha-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the alpha-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by approximately 50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.

SNP haplotypes of the BADH1 gene and their association with aroma in rice (Oryza sativa L.)

Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the synthesis of glycinebetaine—a powerful osmoprotectant against salt and drought stress in a large number of species. Rice is not known to accumulate glycinebetaine but it has two functional genes coding for the BADH enzyme. A non-functional allele of the BADH2 gene located on chromosome 8 is a major factor associated with rice aroma. However, similar information is not available regarding the BADH1 gene located on chromosome 4 despite the similar biochemical function of the two genes. Here we report on the discovery and validation of SNPs in the BADH1 gene by re-sequencing of diverse rice varieties differing in aroma and salt tolerance. There were 17 SNPs in introns with an average density of one per 171 bp, but only three SNPs in exons at a density of one per 505 bp. Each of the three exonic SNPs led to changes in amino acids with functional significance. Multiplex SNP assays were used for genotyping of 127 diverse rice varieties and landraces. In total 15 SNP haplotypes were identified but only four of these, corresponding to two protein haplotypes, were common, representing more than 85% of the cultivars. Determination of population structure using 54 random SNPs classified the varieties into two groups broadly corresponding to indica and japonica cultivar groups, aromatic varieties clustering with the japonica group. There was no association between salt tolerance and the common BADH1 haplotypes, but aromatic varieties showed specific association with a BADH1 protein haplotype (PH2) having lysine144 to asparagine144 and lysine345 to glutamine345 substitutions. Protein modeling and ligand docking studies show that these two substitutions lead to reduction in the substrate binding capacity of the BADH1 enzyme towards gamma-aminobutyraldehyde (GABald), which is a precursor of the major aroma compound 2-acetyl-1-pyrroline (2-AP). This association requires further validation in segregating populations for potential utilization in the rice breeding programs.

Homology Modeling and Molecular Dynamics Simulation Studies of the human resistin protein

Resistin is a member of a secretory protein family, known as resistin-like molecules (RELMs) which were exclusively found in mammalian genomes. Though human resistin molecule has high sequence similarity with mouse, its structural and physiological roles differ considerably from mouse and the structural basis of this drastic functional diffrences are unknown The general objectives of our work are to address these issues and to progress towards a better understanding of the structural basis of resistin biology. Our primary focus is (i) to understand the similarities and differences between human and mouse resistin with respect to their structures and (ii) to infer, using computational approaches, putative functionally important residues. For that purpose we have applied known homology modelling approaches to build a comprehensive 3D model for human resistin using mouse crystallographic data as template. We further assessed the structural properties of this 3D model using molecular dynamics techniques. We importantly compared the properties of both mouse and human resistin structures. The structural status of conserved and non-conserved residues between mouse and human resistin were further investigated with particular emphasis on those residues involved in inter-chain contacts and those exposed on the surface. By identifying the few important residues from the above analysis, we further studied and compared the dynamic properties which provide important insights into structural and functional properties of resistin. Our work suggests that there are considerable differences in interchan interactions and contact surface area between human and mouse structures. Our work also suggests that considerable differences in N-terminal helical orientation in the human model.

Chemical and Structural Probing of the N-Terminal Residues Encoded by FMR1 Exon 15 and Their Effect on Downstream Arginine Methylation

Exon 15 of the fragile X mental retardation protein gene (FMR1) is alternatively spliced into three variants. The amino acids encoded by the 5' end of the exon contain several regulatory determinants including phosphorylation sites and a potential conformational switch. Residues encoded by the 3' end of the exon specify FMRP's RGG box, an RNA binding domain that interacts with G-quartet motifs. Previous studies demonstrated that the exon 15-encoded N-terminal residues influence the extent of arginine methylation, independent of S 500 phosphorylation. In the present study we focus on the role the putative conformational switch plays in arginine methylation. Chemical and structural probing of Ex15 alternatively spliced variant proteins and several mutants leads to the following conclusions: Ex15c resides largely in a conformation that is refractory toward methylation; however, it can be methylated by supplementing extracts with recombinant PRMT1 or PRMT3. Protein modeling studies reveal that the RG-rich region is part of a three to four strand antiparallel beta-sheet, which in other RNA binding proteins functions as a platform for nucleic acid interactions. In the Ex15c variant the first strand of this sheet is truncated, and this significantly perturbs the side-chain conformations of the arginine residues in the RG-rich region. Mutating R 507 in the conformational switch to K also truncates the first strand of the beta-sheet, and corresponding decreases in in vitro methylation were found for this and R 507/R 544 and R 507/R 546 double mutants. These effects are not due to the loss of R 507 methylation as a conformational switch-containing peptide reacted under substrate excess and in methyl donor excess was not significantly methylated. Consistent with this, similar changes in beta-sheet structure and decreases in in vitro methylation were observed with a W 513-K mutant. These data support a novel model for FMRP arginine methylation and a role for conformational switch residues in arginine modification.

The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues

The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed. The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.

Neisseria gonorrheae O-Acetylpeptidoglycan Esterase, a Serine Esterase with a Ser-His-Asp Catalytic Triad

O-Acetylpeptidoglycan esterase from Neisseria gonorrheae FA1090 is similar in sequence to family CE-3 carbohydrate esterases of the CAZy classification system, and it functions to release O-linked acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan. Here, we characterize the peptidoglycan of N. gonorrheae FA1090 as being O-acetylated and find that it serves as a substrate for the esterase. The influence of pH on the activity of O-acetylpeptidoglycan esterase was determined, and pKa values of 6.38 and 6.78 for the enzyme-substrate complex (VEt-1) and free enzyme (VEt-1KM-1), respectively, were calculated. The enzyme was inactivated by sulfonyl fluorides but not by EDTA. Multiple-sequence alignment of the O-acetylpeptidoglycan esterase family 1 enzymes with members of the CE-3 enzymes and protein modeling studies identified Ser80, Asp366, and His369 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of each with alanine was accomplished by site-directed mutagenesis, and the resulting mutant proteins were purified to apparent homogeneity. The specific activity of each of the three esterase derivatives was greatly reduced on O-acetylpeptidoglycan. Using the artificial substrate p-nitrophenyl acetate, a kinetic analysis revealed that the turnover number (VEt-1) but not KM was affected by the replacements. These data thus indicate that N. gonorrheae O-acetylpeptidoglycan esterase, and by analogy the CE-3 family of enzymes, function as serine esterases involving a Ser-His-Asp catalytic triad.

Identification and molecular modelling of a novel familial mutation in the SRY gene implicated in the pure gonadal dysgenesis

SRY gene is responsible for initiating male sexual differentiation. The protein encoded by SRY contains a homeobox (HMG) domain, which is a DNA-binding domain. Mutations of the SRY gene are reported to be associated with XY pure gonadal dysgenesis. The majority of these are de novo mutations affecting only one individual in a family. Only a small subset of mutations is shared between the father and one or more of his children. Most of these familial mutations are localized within the HMG box and only two are at the N-terminal domain of the SRY protein. Herein, we describe a young girl with pure gonadal dysgenesis and her father carrying a novel familial mutation in the SRY gene at codon number 3. This mutation is resulting in a serine (S) to leucine (L) substitution. The secondary structure of the SRY protein was carried out by protein modelling studies. This analysis suggests, with high possibility, that the N-terminal domain of the SRY protein, where we found the mutation, could form an alpha-helix from amino acid in position 2 to amino acid in position 13. The secondary structure prediction and the chemical properties of serine to leucine substitution stands for a potential disruption of this N-terminal alpha-helix in the SRY protein. This mutation could have some role in impeding the normal function of the SRY protein.