Characterization of the N-terminal region within the alkyl hydroperoxide reductase AhpF protein

Cullin3 (Cul3) is an ubiquitin E3 ligase responsible for catalyzing the transfer of an ubiquitin moiety from an E2 enzyme to a target substrate protein. The C-terminal region of Cul3 binds RBX1/E2-ubiquitin, while, the N-terminal region interacts with various BTB domain proteins which serve as substrate adaptors. Previously, our group determined the crystal structures of the homodimeric BTB proteins SPOP and KLHL3 in complex with the N-terminal domain of Cul3, revealing the determinants responsible for the BTB/Cul3 interaction [1, 2]. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors but these do not share many of the previously determined features for Cul3 binding. Furthermore, KCTD proteins form homotetramers and homopentamers via BTB oligomerization rather than the previously described homodimers. Despite these differences, many KCTD proteins interact with Cul3 with dissociation constants of approximately 50 nM. While the target substrates for many of the KCTD/Cul3 E3 ligase complexes are unknown, recent studies have implicated the GABAβ2 receptor as an interactor of KCTD 8, 12, 12b and 16. Here, we report the pentameric crystal structure of the KCTD9 BTB domain and our progress on the structural characterization of Cul3/KCTD/substrate complexes.

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Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

Journal of Bacteriology ◽

10.1128/jb.187.15.5067-5074.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5067-5074 ◽

Cited By ~ 51

Author(s):

Daisuke Kasai ◽

Eiji Masai ◽

Keisuke Miyauchi ◽

Yoshihiro Katayama ◽

Masao Fukuda

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Terminal Region ◽

Ring Cleavage ◽

Sphingomonas Paucimobilis ◽

Acid Protein ◽

Demethylation Pathway ◽

Dioxygenase Gene ◽

Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

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Characterization of antral gastrin cells with region-specific antisera.

Journal of Histochemistry & Cytochemistry ◽

10.1177/25.12.925341 ◽

1977 ◽

Vol 25 (12) ◽

pp. 1317-1321 ◽

Cited By ~ 61

Author(s):

L I Larsson ◽

J F Rehfeld

Keyword(s):

Cross Reactivity ◽

Terminal Region ◽

Gastrin Cells ◽

Related Peptide ◽

Specific Antisera

A number of gastrin antisera, which in radioimmunoassay systems showed no or negligible cross-reactivity towards the structurally and functionally related peptide cholecystokinin were found to react with both gastrin and cholecystokinin cells when used for immunocytochemistry. This discrepancy was shown to be due either to reactivity against a COOH-terminal region common to gastrin and cholecystokinin or to the occurrence of heterogenous antibody populations in the antisera. By differential absorptions the latter type of antisera could be rendered specific for gastrin. Antisera reactive against the NH2-terminal, middle or COOH-terminal regions of human heptadecapeptide gastrin were prepared and together with a specific cholecystokinin antiserum used for the characterization of antral gastrin cells of different species. The results indicate that only the COOH-terminal region of gastrin is conserved during evolution.

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