Optimization of Expression of Human Sulfite Oxidase and Its Molybdenum Domain

Temple, Carrie A.; Graf, Tyler N.; Rajagopalan, K.V.

doi:10.1006/abbi.2000.2089

Cited by 112 publications

(163 citation statements)

References 15 publications

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“…Human sulfite oxidase was purified after the method described in ref. 16, and activity was assayed by monitoring the reduction of cytochrome c at 550 nm.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

Matthies

Rajagopalan

Mendel

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

126

149

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Recent studies have identified the human genes involved in the biosynthesis of the molybdenum cofactor. The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C-terminal segment displaying similarities to the sulfurtransferase rhodanese. The MOCS3 protein is believed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of the smaller subunit of molybdopterin (MPT) synthase. The MOCS3 rhodanese-like domain (MOCS3-RLD) was purified after heterologous expression in E. coli and was shown to catalyze the transfer of sulfur from thiosulfate to cyanide. In a defined in vitro system for the generation of MPT from precursor Z, the sulfurated form of MOCS3-RLD was able to provide the sulfur for the thiocarboxylation of MOCS2A, the small MPT synthase subunit in humans. Mutation of the putative persulfide-forming active-site cysteine residue C412 abolished the sulfurtransferase activity of MOCS3-RLD completely, showing the importance of this cysteine residue for catalysis. In contrast to other mammalian rhodaneses, which are mostly localized within mitochondria, MOCS3 in addition to the subunits of MPT synthase are localized in the cytosol.

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“…Human sulfite oxidase was purified after the method described in ref. 16, and activity was assayed by monitoring the reduction of cytochrome c at 550 nm.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

Matthies

Rajagopalan

Mendel

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

126

149

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“…E. coli BL21(DE3) cells and pET15b were obtained from Novagen. The expression plasmid pTrcHis used for heterologous expression of R. capsulatus XDH in E. coli was described previously (12). For expression of pET15b-based plasmids, the DE3 lysogenization kit from Novagen was used to integrate the gene for T7 RNA polymerase into the E. coli strain RK4353 (11).…”

Section: Methodsmentioning

confidence: 99%

“…The published gene sequence (6) was used to design primers that permitted cloning into the NdeI and HindIII sites of the expression vector pTrcHis (12). The resulting plasmid, designated pSL207, contains the xdh genes with a His 6 tag fused to the N terminus of XDHA.…”

Section: Methodsmentioning

confidence: 99%

“…The conditions for the heterologous expression of R. capsulatus XDH described here were the same as reported for the expression of human sulfite oxidase in E. coli (12). The protein was purified from an 8-liter E. coli culture by Ni-NTA chromatography, followed by chromatography on Q-Sepharose and phenyl-Sepharose (Table I).…”

Section: Heterologous Expression and Purification Of R Capsulatus Xdmentioning

confidence: 99%

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Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

Leimkühler

Hodson

George

et al. 2003

Journal of Biological Chemistry

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Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an (␣␤) 2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds. We have developed an efficient system for the recombinant expression of R. capsulatus XDH in Escherichia coli. The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase. However, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase. X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten L III -edge have been used to probe the different metal coordinations of variant forms of the enzyme. Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Two different forms of XDH-R135C were purified, an active (␣␤) 2 heterotetrameric form and an inactive (␣␤) heterodimeric form. The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.Xanthine dehydrogenase/oxidase (XDH 1 /XO) is a complex metallo-flavoprotein catalyzing the oxidative hydroxylation of purines, pyrimidines, pterines, and aldehyde substrates using NAD ϩ or molecular oxygen as electron acceptor. Mammalian XDH (EC 1.1.1.204) is a homodimer (290 kDa), with each subunit containing a single molybdenum cofactor (Moco) as well as two iron-sulfur clusters and an FAD molecule. Inherited XDH deficiency, referred to as xanthinuria I, is an autosomal recessive disease that is characterized by hyperuricemia, hyperuricosuria, and xanthinuria and is based on base pair exchanges in the structural gene for XDH (1). The affected individuals may develop urinary tract calculi, acute renal failure, or myositis, because of tissue deposition of xanthine, although some subjects with homozygous xanthinuria remain asymptomatic (1). Several mutations in the human XDH gene in patients with classical xanthinuria have been reported (2-4).In their native form, mammalian xanthine oxidizing enzymes exist as dehydrogenases that transfer electrons to NAD but can undergo a dehydrogenase to oxidase conversion by proteolytic cleavage or by oxidation, with the concomitant loss of the ability to use NAD ϩ as electron acceptor, and nearly 10-fold increase in the oxidase activity. The crystal structures of bovine milk XDH in both the dehydrogenase and oxidase forms have been solved at 2.1 and 2.5 Å resolution, respectively (5). It was shown that during the conversion of XDH to XO, a specific structural rearrangement blocks access of NAD ϩ to its binding site near the FAD moiety and changes ...

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“…MOCS2A expression constructs for genetic complementation of the E. coli moaD mutant were cloned into pTrc-His (22). PCR primers were designed to allow cloning of MOCS2A or MocS2B into the NdeI and BamHI sites of the multiple cloning region of pTrc-His to generate pSL203 and pSL173, respectively.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic Studies of Human Molybdopterin Synthase Reaction and Characterization of Mutants Identified in Group B Patients of Molybdenum Cofactor Deficiency

Leimkühler

Freuer

Araujo

et al. 2003

Journal of Biological Chemistry

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Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z.

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Optimization of Expression of Human Sulfite Oxidase and Its Molybdenum Domain

Cited by 112 publications

References 15 publications

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

Recombinant Rhodobacter capsulatus Xanthine Dehydrogenase, a Useful Model System for the Characterization of Protein Variants Leading to Xanthinuria I in Humans

Mechanistic Studies of Human Molybdopterin Synthase Reaction and Characterization of Mutants Identified in Group B Patients of Molybdenum Cofactor Deficiency

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