A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), beta-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.

SummaryBackground and objectives Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1a,25(OH) 2 D 3 ) in the development of those disorders in two individuals.Design, setting, participants, & measurements Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis.Results Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1a,25(OH) 2 D 3 ), normal 25-OHD 3 , decreased 24,25(OH) 2 D, and undetectable activity of 1,25(OH) 2 D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1a,25(OH) 2 D 3 . Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%-20%. ConclusionsThe results of this study show that 1,25(OH) 2 D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

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ABSTRACT:Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k cat /K m value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3) and 25-hydroxyvitamin D 3 (25(OH)D 3) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH) 2 D 3 with an affinity equivalent to that for 25(OH)D 3 , were also generated. Although Cyp27b1-knockout (KO), Vdr-Ko, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D 3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH) 2 D 3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D 3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D 3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system. The active form of vitamin D 3 , 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3), plays important roles in osteogenesis, calcium homeostasis, cellular differentiation, and immune responses 1. 1,25(OH) 2 D 3 is generated by two hydroxylation steps from vitamin D 3 : C-25 hydroxylation by hepatic CYP2R1 and CYP27A1 and subsequent 1α-hydroxylation by renal 1α-hydroxylase (CYP27B1) 2. The vitamin D receptor (VDR) mediates the genomic action of active vitamin D 3. Binding of active vitamin D 3 to the VDR triggers its heterodimerization to the retinoid X receptor and subsequent translocation to the nucleus. This translocation results in regulating target gene expression by formation of the VDR complex on vitamin D-responsive elements in the promoter regions of target genes, such as osteocalcin and osteopontin in bones and the calcium channels and calbindins in intestines 3. CYP24A1, one of the well-known vitamin D target genes, is involved in inactivating 1,25(OH) 2 D 3 through sequential metabolism that starts with C-24 or C-23 hydroxylation of 1,25(OH) 2 D 3 4. A variety of vitamin D derivatives have been developed as drugs for rickets, osteoporosis, psoriasis, secondary hyperparathyroidism, and chronic kidney disease. Because all of these compounds show high affinity for the VDR, these pharmacological actions are considered to be VDR mediated. However, as with 1,25(OH) 2 D 3 , they might also have non-VDR-mediated actions. Thus, pharmacological action studies of vitamin D derivatives are essential for future drug discovery.