Preparation of Crystalline Phosphorylated Derivatives of Vitamin B<sub>6</sub>

Peterson, Elbert A.; Sober, Herbert A.

doi:10.1021/ja01630a045

Cited by 493 publications

(165 citation statements)

References 2 publications

Supporting

Mentioning

161

Contrasting

Unclassified

Order By: Relevance

“…N-His 6 -NocJ was treated with sodium borohydride, (37) resulting in loss of the peak at 420 nm, and the formation of a new band at 325 nm, consistent with reduction of the imine bond of bound PLP (data not shown) (38). By obtaining the UV-visible spectrum of N-His 6 -NocJ in 0.1 N sodium hydroxide (⑀ 288 ϭ 6600 M Ϫ1 cm Ϫ1 for PLP), (23) it was determined 1 mol of NocJ binds 1.4 mol of PLP.…”

Section: Analysis Of Nocj-mentioning

confidence: 90%

See 1 more Smart Citation

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic ␤-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a D-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5 -phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the L-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9 epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of ␤-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.

show abstract

Section: Analysis Of Nocj-mentioning

confidence: 90%

“…Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard (22). The pyridoxal 5Ј-phosphate content of NocJ was determined by obtaining the optical spectrum of the protein in 0.1 N NaOH using ⑀ 388 of 6600 M Ϫ1 cm Ϫ1 (23).…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Nfs1⌬1-55 concentrations were determined from the absorbance at 420 nm using the extinction coefficient of 10.9 mM Ϫ1 cm Ϫ1 for the native enzyme. The extinction coefficient was determined on the basis of the PLP content after alkaline denaturation described after the method of Peterson and Sober (30).…”

Section: Methodsmentioning

confidence: 99%

A Novel Role for Human Nfs1 in the Cytoplasm

Marelja

Stöcklein

Nimtz

et al. 2008

Journal of Biological Chemistry

111

View full text Add to dashboard Cite

The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an L-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human L-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from L-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.

show abstract

“…Pyridoxal 5'-P and pyridoxamine 5'-P, which were chromatographically purified by the method of Peterson and Sober [8], were products of Kyowa Hakko Kogyo, Tokyo. Sephadex G-25 was obtained from Pharmacia, Uppsala.…”

Section: Methodsmentioning

confidence: 99%