The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues

Blake, C. C. F.; Ghosh, Minakshi; Harlos, K.; Avezoux, A; Anthony, Christopher

doi:10.1038/nsb0294-102

Cited by 104 publications

(87 citation statements)

References 21 publications

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“…The first conformational analysis of the oxidized Cys-Cys dipeptide suggested that the ring might lead to a turn when found in the middle of a peptide chain (20)(21)(22)(23)(24)(25). The analysis of model peptides confirmed that the formation of the vicinal disulfide bond is accompanied by the appearance of a tight ␤-type turn, and this phenomenon is also used in rational peptide design (26)(27)(28)(29).…”

Section: Resultsmentioning

confidence: 87%

“…It occurs in quite unrelated proteins such as the J-Atracotoxin from Hadronyche versuta (26), methanol dehydrogenase from Methylobacterium extorquens (27)(28)(29), the ethanol dehydrogenase from Pseudomonas aeruginosa (30), acetylcholine-binding protein from Lymnaea stagnalis (31), thioesterase I from Bos taurus (32), carboxypeptidase T from Thermoacinomyces vulgaris (33), and antimicrobial peptide hepcidin-25 from Homo sapiens (34) (Fig. 4 A-G).…”

Section: Resultsmentioning

confidence: 99%

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Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Cemazar

Zahariev

Lopez

et al. 2003

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

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The oxidative folding of the Amaranthus ␣-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by 1 H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.T he cystine knot, or the knottin fold, is a compact structure consisting of a short triple-stranded antiparallel ␤-sheet reinforced by three disulfide bridges that form a topological knot [see Amaranthus ␣-amylase inhibitor (AAI) structure, Fig. 1A Inset]. It is found both in small peptides and as a domain of larger proteins (1) and in addition, it is a recurrent substructure of larger cysteine-rich motifs such as the well known conotoxin fold (2). Despite their small size, cystine knots can fulfill a large variety of functions ranging from ion-channel blocking to enzyme inhibition, which makes them ideal protein engineering scaffolds for industrial applications (3, 4). Because cystine knots occur in many unrelated species (fungi, plants, snails, and spiders, etc.), it is presumed that this fold has emerged via convergent evolution (2). The compactness and versatility of this fold are best illustrated by the fact that both the shortest peptide inhibitor of ␣-amylases, AAI [32 amino acids (Fig. 1 A)] (5-8) and the shortest lectin molecule known so far (9), are members of this family.Cystine knot peptides are known to form readily in vitro under the conditions of oxidative folding (4). In this work, we describe an analysis of the oxidative folding intermediates of AAI, which shows that the native protein (N) forms via several fully oxidized intermediates with nonnative disulfide bonds, including the vicinal disulfide 17-18. NMR spectroscopy revealed that the main folding intermediate has a structure as compact as the completely folded peptide, comprising a number of slowly interconverting backbone conformations. On the basis of the experimental data, we suggest that the compactness, a prerequisi...

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Cemazar

Zahariev

Lopez

et al. 2003

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

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“…A disulfide bond between adjacent cysteines joined by a cis-peptide bond has been rarely reported, i.e. only for methanol dehydrogenase (39) and human ribonuclease inhibitor (40). In methanol dehydrogenase from Methylobacterium extorquens, the disulfide bond formed by Cys 103 and Cys 104 lies immediately above the pyrroloquinoline quinone.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of a Family 54 α-l-Arabinofuranosidase Reveals a Novel Carbohydrate-binding Module That Can Bind Arabinose

Miyanaga

Koseki

Matsuzawa

et al. 2004

Journal of Biological Chemistry

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As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 ␣-L-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a ␤-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a ␤-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/ base residues were determined to be Glu 221 and Asp 297 , respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.

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“…Such an atypical disulfide bridge, connecting residues that are adjacent in sequence, is rare in available protein structures. Two of these proteins are members of the alcohol dehydrogenase family, specifically methanol dehydrogenase from Methylobacterium extorquens (48,49) and ethanol dehydrogenase from Pseudomonas aeruginosa (50). The atypical disulfide bridge may stabilize the non-planar semiquinone form of the enzyme's prosthetic group pyrroloquinoline quinone (49).…”

Section: Zinc Is a Competitive Active Site Inhibitor That Binds To Thementioning

confidence: 99%

Differential Regulation of a Hyperthermophilic α-Amylase with a Novel (Ca,Zn) Two-metal Center by Zinc

Lindén

Mayans

Meyer‐Klaucke

et al. 2003

Journal of Biological Chemistry

102

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The crystal structure of the ␣-amylase from the hyperthermophilic archaeon Pyrococcus woesei was solved in the presence of three inhibitors: acarbose, Tris, and zinc. In the absence of exogenous metals, this ␣-amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, twometal (Ca,Zn)-binding site and a second inhibitory zincbinding site that is found in the ؊1 sugar-binding pocket within the active site. The data resolve the apparent paradox between the zinc requirement for catalytic activity and its strong inhibitory effect when added in molar excess. They provide a rationale as to why this ␣-amylase, in contrast to commercially available ␣-amylases, does not require the addition of metal ions for full catalytic activity, suggesting it as an ideal target to maximize the efficiency of industrial processes like liquefaction of starch.

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The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues

Cited by 104 publications

References 21 publications

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Crystal Structure of a Family 54 α-l-Arabinofuranosidase Reveals a Novel Carbohydrate-binding Module That Can Bind Arabinose

Differential Regulation of a Hyperthermophilic α-Amylase with a Novel (Ca,Zn) Two-metal Center by Zinc

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