Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family.
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ABSTRACT:
SUBMITTER: Dolphin CT
PROVIDER: S-EPMC1133153 | biostudies-other | 1992 Oct
REPOSITORIES: biostudies-other
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Project description:AimThe aim of this study was to investigate intra- and inter-individual variations of functional metabolic capacity of flavin-containing mono-oxygenase (FMO) during childhood using trimethylamine N-oxygenation as a probe reaction.MethodsTrimethylamine N-oxygenation functional activity and presence of FMO1 (fetal form), FMO3 (adult form), and FMO5 (endogenous form) were immunochemically determined and compared in human liver microsomes obtained from children at various ages. As a control, the same parameters were studied with recombinant FMO1, FMO3 and FMO5 proteins as enzyme sources. Developmental variation in functional metabolic capacity of FMO was estimated by measuring urinary trimethylamine and its N-oxide in several individuals at different ages and in a group of 77 subjects in childhood.ResultsThere was a significant correlation between trimethylamine N-oxygenation functional activity and FMO3 expression levels in human liver microsomes (r= 0.71, P < 0.05, n= 9). Trimethylamine N-oxygenation was catalyzed largely by FMO3 and not by FMO1 or FMO5. On the basis of analysis of intra-individual observations and collective urine samples under daily dietary conditions it was possible that neonates or infants harbouring at least one non-inactive-allele of the FMO3 gene could have developmental FMO3 metabolic capacity in childhood.ConclusionsDevelopmental variations in functional metabolic capacity of FMO3 in childhood were shown both on the basis of in vivo phenotyping tests and in in vitro liver microsomal determinations.
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Project description:AIM: The aim of this study was to investigate relationships between flavin-containing mono-oxygenase 3 (FMO3) genotype and phenotype (conversion of odorous trimethylamine into non-odorous trimethylamine N-oxide) in a large Japanese cohort suffering from trimethylaminuria. METHODS: Urinary excretion of trimethylamine and trimethylamine N-oxide was determined for 102 volunteers with self-reporting symptoms of trimethylaminuria. For each we determined the sequence of the entire coding region, plus 1.3?kb of flanking intronic and 2.5?kb of the upstream region of the FMO3 gene. The affect of upstream variants on transcription was determined with a reporter gene assay. RESULTS: Seventy-eight subjects were diagnosed as suffering from trimethylaminuria, based on urinary excretion of <90% of total TMA as TMA?N-oxide. Of these, 13 were classified as severe, 56 as moderate and nine as mild cases, excreting <43%, 48-70% and 73-83% of trimethylamine as trimethylamine N-oxide, respectively. Twenty-seven mutations were identified in FMO3, 15 in the coding region, of which eight abolish or severely impair FMO3 activity (Pro70Leu, Cys197fsX, Thr201Lys, Arg205Cys, Met260Val, Trp388Ter, Gln470Ter and Arg500Ter), and 12 in the upstream region. The mutations segregate into 19 haplotypes, including four different combinations of upstream mutations, each of which reduces transcriptional activity in comparison with the ancestral upstream sequence of FMO3. CONCLUSIONS: Comparisons of genotype and phenotype reveal that severe trimethylaminuria is caused by loss of function mutations in FMO3. For moderate and mild cases the situation is more complex, with most resulting from factors other than FMO3 genotype. Our results have implications for the diagnosis and management of the disorder.
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Project description:BACKGROUND:Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. RESULTS:Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. CONCLUSIONS:Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.
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Project description:The mechanisms that determine the relative positions of floral organs, and thereby their numbers, is a poorly understood aspect of flower development. We isolated a petunia mutant, floozy (fzy), in which the formation of floral organ primordia in the outermost three floral whorls and one of the two bracts at the base of the flower is blocked at an early stage. In addition, fzy mutants fail to generate secondary veins in leaves and bracts and display a decreased apical dominance in the inflorescence. FZY encodes an enzyme with homology to flavin mono-oxygenases and appears to be the ortholog of YUCCA genes of Arabidopsis. FZY is expressed in young leafs and bracts and in developing flowers. In young floral meristems FZY is expressed in the center of the meristem dome and, later, expression becomes localized on the flanks of the initiating petal and stamen primordia and at several sites in maturing anthers and carpels. These findings indicate that FZY is involved in synthesizing a signaling compound that is required for floral organ initiation and specification of the vascularization pattern in leaves. Although fzy mutants contain normal auxin levels, ectopic expression of FZY results in excessive auxin accumulation, suggesting that the signaling compound is auxin.
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