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PubMed2026

Translational case series comparing next-generation sequencing profiles of primary breast cancer and brain metastases.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease, and its molecular and immunohistochemical (IHC) profiles may change over time, particularly under therapeutic pressure. IHC discordance between primary tumors and BC brain metastases (BCBM) has been reported, yet its biological and clinical significance remains incompletely defined. Genomic profiling using next-generation sequencing (NGS) may provide additional insight into tumor evolution and clonal selection, although data from paired BC and BCBM are limited. METHODS: This translational case series included six patients randomly selected from an institutional cohort of BC patients who underwent neurosurgical resection of BCBM. IHC reassessment (ER, PR, and HER2) and NGS profiling using targeted panels were performed. RESULTS: Three of the six cases presented with IHC discordance, mainly loss of HR expression and gain of HER2 in BCBM. Genomic profiling identified 23 mutations in primary tumors compared with four in BCBM. BRCA1/2 variants predominated in primary tumors (21/23, 91%), most predicted to result in loss-of-function alterations. One mutation (PIK3CA/N345K) was shared between primary and metastatic tissues within the same patient. Overall survival ranged from 28 to 146 months. CONCLUSION: This paired analysis demonstrates immunophenotypic and genomic divergence between BC and BCBM, supporting the concept of dynamic tumor evolution. Receptor conversion and emergence or loss of actionable genomic alterations highlight the potential value of repeat molecular assessment in advanced stages. Although limited by a small sample size, retrospective design, and absence of matched germline testing, these findings reinforce the importance of integrating biomarker reevaluation into the management of selected patients.

Humans↗

PubMed2004

Mutagenesis at the human tachykinin NK(2) receptor to define the binding site of a novel class of antagonists.

The pharmacological profile of novel antagonists endowed with high affinity for the human tachykinin NK(2) receptor is presented. MEN13918 (Ngamma[Nalpha[Nalpha(benzo[b]thiophen-2-yl)carbonyl]-1-aminocyclohexan-1-carboxy]-d-phenylalanyl]-3-cis-aminocyclohexan-1-carboxylic-acid-N-(1S,2R)-2-aminocyclohexyl)amide trifluoroacetate salt) and MEN14268 (Nalpha[Nalpha(benzo[b]thiophen-2-yl)carbonyl)-1-aminocyclopentane-1-carboxyl]-d-phenylalanine-N-[3(morpholin-4-yl)propyl]amide trifluoroacetate salt) were more potent in blocking neurokinin A (NKA, His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH(2)) induced contraction in human, which induced greater contraction in human (pK(B) 9.1 and 8.3) than rat (pK(B) 6.8 and <6) urinary bladder smooth muscle preparation in vitro. In agreement with functional data, in membrane preparations of CHO cells stably expressing the human NK(2) receptors, both MEN13918 and MEN14268 potently inhibited the binding of agonist ([(125)I]NKA, K(i) 0.2 and 2.8 nM) and antagonist ([(3)H]nepadutant, K(i) 0.1 and 2.2 nM, [(3)H]SR48968 K(i) 0.4 and 6.9 nM) radioligands. Using site-directed mutagenesis and radioligands binding we identified six residues in the transmembrane (TM) helices that are critical determinants for the studied antagonists affinity. To visualize these experimental findings, we constructed a homology model based on the X-ray crystal structure of bovine rhodopsin and suggested a possible binding mode of these newly discovered antagonist ligands to the human tackykinin NK(2) receptor. Both MEN13918 and MEN14268 bind amongst TM4 (Cys167Gly), TM5 (Tyr206Ala), TM6 (Tyr266Ala, Phe270Ala), and TM7 (Tyr289Phe, Tyr289Thr). MEN13918 and MEN14268 diverging binding profile at Y289 mutations in TM7 (Tyr289Phe, Tyr289Thr) suggests a relation of their different chemical moieties with this residue. Moreover, the different influence on binding of these two ligands by mutations located deep along the inner side of TM6 (Phe270Ala, Tyr266Ala, Trp263Ala) indicates a nonequivalent positioning, although occupying the same binding crevice. Furthermore, binding data indicate the Ile202Phe mutation, which mimics the wild-type rat NK(2) receptor sequence, as a species selectivity determinant. In summary, data with mutant receptors describe, for these new tachykinin NK(2) receptor antagonists, a binding site which is partially overlapping either with that of the cyclized peptide antagonist nepadutant (cyclo-[[Asn(beta-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)] or the nonpeptide antagonist SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide).

Animals↗

PubMed2000

Prevalence of point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum isolates from India and Thailand: a molecular epidemiologic study.

Pyrimethamine-sulfadoxine (PS) is used as a second-line treatment for P. falciparum malaria patients who fail to respond to chloroquine. Resistance to these drugs has been shown to encode with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes. Our aim was to assess the comparative rate of point mutation occurring in DHFR and DHPS genes among P. falciparum isolates from India and Thailand where the use of PS is at a different rate. We used the mutation-specific polymerase chain reaction (PCR) technique and mutation-specific restriction digestion to determine the prevalence of DHFR and DHPS gene mutations at codons 16, 51, 59, 108, 164 and at 436, 437, 581 and 613, respectively. In the 89 clinical isolates from India, in the case of the DHFR gene, we found 71 of S108N, 10 of N51I, 28 of C59R and four of I164L types. Among the 50 isolates from Thailand the rate of point mutations in the DHFR gene was higher at four codon positions. We found 47 of S108N, 18 of N51I, 23 of C59R and 12 of I164L types. None of the isolates from either country possessed the paired mutations S108T and A16V. Mutations of the DHPS gene were less frequent among the Indian isolates: 4.5% showed DHPS gene mutation, two of S436F, A437G, A613T and two of S436F, A613T; whereas 66% (33/50) of the Thai isolates had mutated at codons 436, 437, 581 and 613 which include 13 of S436F, 15 of A437G, 19 of A581G and 25 of A613S/T, ranging from single to quadruple mutant types. Among the Indian isolates, DHFR point mutations were very frequent and 85/89 had a wild type DHPS genetic profile. The pattern of mutations in the samples from Thailand was different, as most were associated with point mutations in DHFR and DHPS genes.

Adolescent↗

PubMed2004

Capravirine, a nonnucleoside reverse-transcriptase inhibitor in patients infected with HIV-1: a phase 1 study.

Capravirine is a nonnucleoside reverse-transcriptase inhibitor (NNRTI) with a unique resistance profile. Although single mutations allow resistance to established NNRTIs, human immunodeficiency virus (HIV)-1 must undergo multiple mutations to achieve resistance to capravirine. In the present phase 1 study, capravirine was administered orally for up to 28 days to 55 HIV-1-infected individuals with CD4+ T lymphocyte counts of 50-500 cells/microL. The most frequent adverse events were diarrhea (5%) and nausea (4%), with no drug-related rashes observed. The day 15 median (mean) HIV-1 load decreased by 1.34 (1.45) log(10) copies/mL in the patients receiving 25 mg/kg/day. Capravirine demonstrated potent antiviral activity, even in antiretroviral-experienced patients.

Administration, Oral↗

PubMed1998

A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana.

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

Amino Acid Sequence↗

PubMed2005

Interaction analysis between tmRNA and SmpB from Thermus thermophilus.

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.

Bacterial Proteins↗

PubMed1999

Establishment and characterization of human laryngeal squamous cell carcinoma cell lines.

OBJECTIVES: Six human laryngeal squamous cell carcinoma cell lines (SNU-46, -585, -899, -1066, -1076, -1214) established from Korean patients are reported. STUDY DESIGN: In vitro culture of six squamous cell carcinoma cell lines derived from primary tumors of the larynx. Description of the cell line phenotypes and determination of molecular characteristics. METHODS: Six laryngeal squamous cell carcinoma cell lines were cultured. The cell phenotypes, including the histopathology of the primary tumors and in vitro growth characteristics, were determined. Molecular characterization was also performed, including DNA fingerprinting analysis and abnormalities of p15, p16, p53, and TGF-betaRII genes by polymerase chain reaction-based single strand conformation polymorphism and sequencing analysis. RESULTS: All cell lines grew as adherent cells; five lines grew as monolayers and one other line grew as stratifying colonies. All lines showed 1) high viability (75%-92%) with various doubling times (36-96 h); 2) absence of Mycoplasma and other bacteria; and 3) genetic heterogeneity by DNA profile analysis. p53 Mutations were found in three lines and p16 mutations were observed in five cell lines. TGF-betaRII mutations were found in two lines: one line had frameshift mutation and another line had a missense mutation at the kinase domain. CONCLUSIONS: These newly established and characterized laryngeal squamous cell carcinoma cell lines will be useful for investigating the biologic characteristics of laryngeal cancer.

Carcinoma, Squamous Cell↗

PubMed2005

Oligodendroglioma: impact of molecular biology on its definition, diagnosis and management.

Molecular genetics and biology have been having significant influence on the practice of neuro-oncology in recent years, with oligodendrogliomas being the most prominent example. The majority of oligodendrogliomas show remarkable sensitivity to chemotherapy, and the finding that the loss of chromosome 1p was tightly associated with the response opened a new era in which the treatment can be tailored for each tumor patient based on molecular genetic diagnosis. It has been noticed that histological features, when closely examined, are also correlated with the molecular genetic data, such as losses of 1p/19q/10q and TP53 mutation. Furthermore, expression profiling using microarray technology showed that oligodendrogliomas with 1p loss express high levels of neuronal genes, suggesting that the progenitor of those tumors may be shared with neurons. Considering the rapid progress of the knowledge in neural developmental biology, it is possible that the classification, definition and diagnosis of gliomas will be rewritten based on such knowledge at the molecular level in the near future, if they reflect the biological features of each tumor better than the histological diagnosis based mostly on morphology.

Animals↗

PubMed1993

TnphoA Salmonella abortusovis mutants unable to adhere to epithelial cells and with reduced virulence in mice.

Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.

Abortion, Veterinary↗

PubMed1992

Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.

Base Sequence↗

PubMed2005

A generic approach for the design of whole-genome oligoarrays, validated for genomotyping, deletion mapping and gene expression analysis on Staphylococcus aureus.

BACKGROUND: DNA microarray technology is widely used to determine the expression levels of thousands of genes in a single experiment, for a broad range of organisms. Optimal design of immobilized nucleic acids has a direct impact on the reliability of microarray results. However, despite small genome size and complexity, prokaryotic organisms are not frequently studied to validate selected bioinformatics approaches. Relying on parameters shown to affect the hybridization of nucleic acids, we designed freely available software and validated experimentally its performance on the bacterial pathogen Staphylococcus aureus. RESULTS: We describe an efficient procedure for selecting 40-60 mer oligonucleotide probes combining optimal thermodynamic properties with high target specificity, suitable for genomic studies of microbial species. The algorithm for filtering probes from extensive oligonucleotides libraries fitting standard thermodynamic criteria includes positional information of predicted target-probe binding regions. This algorithm efficiently selected probes recognizing homologous gene targets across three different sequenced genomes of Staphylococcus aureus. BLAST analysis of the final selection of 5,427 probes yielded >97%, 93%, and 81% of Staphylococcus aureus genome coverage in strains N315, Mu50, and COL, respectively. A manufactured oligoarray including a subset of control Escherichia coli probes was validated for applications in the fields of comparative genomics and molecular epidemiology, mapping of deletion mutations and transcription profiling. CONCLUSION: This generic chip-design process merging sequence information from several related genomes improves genome coverage even in conserved regions.

Algorithms↗

PubMed2006

Clock mutation affects circadian regulation of circulating blood cells.

BACKGROUND: Although the number of circulating immune cells is subject to high-amplitude circadian rhythms, the underlying mechanisms are not fully understood. METHODS: To determine whether intact CLOCK protein is required for the circadian changes in peripheral blood cells, we examined circulating white (WBC) and red (RBC) blood cells in homozygous Clock mutant mice. RESULTS: Daytime increases in total WBC and lymphocytes were suppressed and slightly phase-delayed along with plasma corticosterone levels in Clock mutant mice. The peak RBC rhythm was significantly reduced and phase-advanced in the Clock mutants. Anatomical examination revealed hemoglobin-rich, swollen red spleens in Clock mutant mice, suggesting RBC accumulation. CONCLUSION: Our results suggest that endogenous clock-regulated circadian corticosterone secretion from the adrenal gland is involved in the effect of a Clock mutation on daily profiles of circulating WBC. However, intact CLOCK seems unnecessary for generating the rhythm of corticosterone secretion in mice. Our results also suggest that CLOCK is involved in discharge of RBC from the spleen.

Journal Article↗

PubMed2006

Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain.

Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3) domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders) and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well.

Amino Acid Motifs↗

PubMed2005

Prenatal molecular diagnosis of adrenoleukodystrophy.

OBJECTIVE: To carry out prenatal diagnosis on two fetuses of different pedigrees with X-linked adrenoleukodystrophy (ALD). METHODS: The amniotic fluid was obtained with the help of a clinical doctor and the genomic DNA was isolated from it. Maternal DNA contamination was excluded by fluorescent STR profiling, The R617G mutation found in the first pedigree was searched in genomic DNA of amniotic fluid cells (AFC) from fetus 1 by amplification refractory mutation system (ARMS) and dot DNA hybridization while the P534R mutation found in pedigree 2 was analyzed in the AFC genomic DNA of fetus 2 by restrictive digestion with Hae II and DNA direct sequencing. RESULTS: A specific band (185 bp) was detected from the genomic DNA of the first fetus and his mother by using mutation primer in ARMS but not from that of the first fetus's father and unrelated controls. DNA dots were visualized only in the fetus 1 and carrier when using the mutation probe in DNA hybridization. In the other ALD family, the PCR product (506 bp) of the second fetus which spanned the site of P534R mutation could not be digested with Hae II and no mutation was detected in the ABCD1 gene from the genomic DNA of the fetus 2 by using DNA direct sequencing. CONCLUSION: Fetus 1 had R617G mutation on his ABCD1 gene and he was an adrenoleukodystrophy hemizygote. Fetus 2 had no P534R mutation on his ABCD1 gene and he was a normal hemizygote.

ATP Binding Cassette Transporter, Subfamily D, Mem↗

PubMed2006

[Familial adenomatous polyposis: Gardner's syndrome].

Familial adenomatous polyposis (PAF) associated to soft tissue tumors or osteomas constitutes the Gardner's syndrome of autosomal dominant inheritance. The risk of colorectal cancer in these patients is 100%. We present a patient with Gardner's syndrome who was had colectomy at early age. An eleven years old boy he was evaluated due to a family history of PAF and subcutaneous tumors (occipital and left thigh). Genetic profile shows a mutation in gene APC and the colonoscopy confirms the polyposis; the biopsy also suggested moderate dysplasia. When the patient reached the age of twelve, a total colectomy with colorectal mucosectomy was performed. Cystic subcutaneous lesions (epidermoid cysts) were also excised. In the postoperative period there were no complications. The prophylactic colectomy, is the only effective treatment to prevent the colorectal cancer. Gardner's syndrome patients requires periodic controls to rule out the appearance of new tumors or anomalies in the retine. The duodenoscopy is essential in the follow up of these patients because of the frequency of duodenal affectation.

Adenomatous Polyposis Coli↗

PubMed1994

Heterogeneity of subcellular localization of p53 protein in human glioblastomas.

Immunohistochemical analysis of the p53 protein in human glioblastomas with known genetic profiles of p53 mutations and allele losses on chromosome 17p demonstrated a heterogeneous pattern of subcellular compartmentalization of the p53 protein. Tumors with a single wild type copy of the p53 gene but with allelic deletions on chromosome 17p exhibit nuclear and/or cytoplasmic accumulation of p53, whereas tumors with both copies of the wild type gene and no allele losses on chromosome 17 do not accumulate p53. Glioblastomas with one normal and one mutated copy of the p53 gene and allelic deletions on 17p distal to p53, on the other hand, show predominantly cytoplasmic staining, probably originating from the wild type p53 protein. Furthermore, tumors with mutations in the same codon of p53 display quite different intracellular distribution suggesting that, in addition to the genotype of p53, the intracellular microenvironment of a particular tumor is important in determining the subcellular localization of the p53 protein.

Base Sequence↗

PubMed1997

[Biochemical and genetic mechanisms for bacteria to acquire aminoglycoside antibiotic resistance].

Aminoglycoside (AG)-modifying enzymes are the major biochemical basis for the AG resistance of clinically-occurring bacteria. Recent AG resistance profiles can be characterized by the involvement of AAC(6') in combination with other modifying enzymes in Gram negative bacteria. AAC(6')/APH(2") in Staphylococcus aureus is also remarkable. Genetic basis for the emergence or alteration of AG resistance profiles includes point mutations in the regulatory region or specific sites of the coding region of AG-modifying enzyme genes, and rearrangement of the genes caused by transposon and/or integron. In addition, semisynthetic AG antibiotics such as amikacin, arbekacin (ABK) and isepamicin were also reviewed for their stability to AG-modifying enzymes. ABK that has been widely used as an anti-MRSA drug in Japan is distinct from the other AGs because its monoacetylated derivatives (3"-N-acetylABK and 2'-N-acetylABK) by AG acetyltransferases, AAC(3) and AAC(2'), respectively, retain clear antibiotic activities. Based on this novel aspect and the lack of modification sites for APH(3') and ANT(4'), ABK should be regarded as the most refractory AG for bacteria to acquire resistance.

Acetyltransferases↗

PubMed1999

Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.

Acetobacter↗