Crystal structure of apo‐glycolate oxidase

Sandalova, T.; Lindqvist, Ylva

doi:10.1016/0014-5793(93)81021-q

Cited by 14 publications

(10 citation statements)

References 31 publications

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“…In such cases conformational changes induced by binding of the hydride acceptor are well documented (27)(28)(29)(30). In a striking example of this, the structure of glycolate oxidase verges on a molten globule-like state in the absence of the co-factor, FMN (30). Binding of the co-factor induces widespread structural reorganization and stabilization, whereupon the enzyme active site residues are oriented optimally for catalysis.…”

Section: Discussionmentioning

confidence: 99%

“…In most other dehydrogenases substrate binding is either random or the hydride acceptor binds first (26). This binding often induces a large conformational change that prepares the enzyme for the catalytic events (27)(28)(29)(30). In the case of IMPDH, catalysis proceeds via an ordered bi-bi kinetic mechanism wherein IMP (the hydride donor) binds first and NAD ϩ binds second.…”

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confidence: 99%

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Conformational Changes and Stabilization of Inosine 5′-Monophosphate Dehydrogenase Associated with Ligand Binding and Inhibition by Mycophenolic Acid

Nimmesgern

Fox

Fleming

et al. 1996

Journal of Biological Chemistry

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The effects of substrate, product, and inhibitor (mycophenolic acid) binding on the conformation and stability of hamster type II inosine 5-monophosphate dehydrogenase (IMPDH) have been examined. The protein in various states of ligand occupancy was compared by analyzing susceptibility to in vitro proteolysis, the degree of binding of a hydrophobic fluorescent dye, secondary structure content as determined by far-UV circular dichroism spectra, and urea-induced denaturation curves. These analysis methods revealed consistent evidence that IMPDH undergoes a local reorganization when IMP or XMP bind. NAD ؉ produced no such effect. In fact, no evidence was found for NAD ؉ binding independently of IMP. It is proposed that IM-PDH adopts an open conformation around its nucleotide binding sites in the absence of substrates and that binding of IMP stabilizes a closed conformation that has a higher affinity for NAD ؉ . The data also suggest the enzyme remains in the closed configuration throughout the catalytic steps and then reverts to the open conformation with XMP release, thereby consummating the enzyme cycle. Mycophenolic acid inhibition appeared to impart even greater stability. We propose that localized conformational changes occur during the normal and mycophenolic acid-inhibited reaction sequences of IMPDH.Inosine 5Ј-monophosphate dehydrogenase (IMPDH; 1 EC 1.1.1.205) catalyzes the conversion of IMP to XMP, the committed step in the de novo biosynthesis of guanine nucleotides (1-4). Now partially characterized from many organisms, IMPDH is well conserved with 35% or more sequence identity from bacterial and protozoan to human proteins (5, 6). Since the enzyme appears to be necessary for cellular replication, some view it as an attractive target for anti-microbial chemotherapy; however, others have recognized further therapeutic possibilities for IMPDH inhibition.Human and hamster possess two closely related isoenzymes (designated types I and II). In both species the isoforms share 84% (of 514 residues) sequence identity (5).2 The existence of similar isoenzymes in mammals suggests a subtle distinction of roles. Many studies have shown that IMPDH activity is elevated in tumors and other actively proliferating tissues (3,4,(7)(8)(9)(10)(11)(12)(13)(14)(15). In some cases the elevated activity seems to stem from induction of the type II isoenzyme (9, 11, 12), although similar up-regulation of both isoforms in activated T-lymphocytes has also been reported (14). These observations suggest that inhibition of IMPDH, or perhaps selective inhibition of individual isoforms, has potential for immunosuppressive or anti-neoplastic therapies. Indeed, inhibition of IMPDH in cultured cells results in decreased guanine nucleotide levels. Ultimately, this leads to the induction of differentiation in neoplastic cells (8) and the suppression of lymphocyte activation (16).Mycophenolic acid (MPA), an uncompetitive inhibitor of IMPDH that was originally identified in certain Penicillium cultures (17), is a potent immunosuppressant, bot...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Conformational Changes and Stabilization of Inosine 5′-Monophosphate Dehydrogenase Associated with Ligand Binding and Inhibition by Mycophenolic Acid

Nimmesgern

Fox

Fleming

et al. 1996

Journal of Biological Chemistry

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“…18 In ThDP-dependent enzymes, it has been reported that TK shows a disorder -order transition of two loop regions upon binding of the cofactors ThDP and Ca 2þ . 16 However, it is unclear whether or not this is a common feature of ThDP-dependent enzymes, since structures of other ThDP-dependent enzymes in both apo and holo-forms have not been elucidated.…”

Section: Disorder-order Transition Upon Binding Of the Cofactors Thdpmentioning

confidence: 99%

Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

Nakai

Nakagawa

Maoka

et al. 2004

Journal of Molecular Biology

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“…Flavoproteins represent one of the major groups of cofactordependent enzymes (1,2). However, data on the molecular process of FAD or FMN binding are scarce, while only a limited number of flavoprotein structures in the apo form are known (3)(4)(5). A striking example of the importance of efficient FAD binding was recently shown for methylenetetrahydrofolate reductase (6).…”

mentioning

confidence: 99%

Structural Analysis of Flavinylation in Vanillyl-Alcohol Oxidase

Fraaije¹,

Heuvel²,

Berkel³

et al. 2000

Journal of Biological Chemistry

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Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 3 Thr). In the mutant enzyme the covalent His-C8␣-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K d ‫؍‬ 1.8 and 2.1 M, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k cat ‫؍‬ 0.24 s ؊1 , K m ‫؍‬ 40 M) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.A substantial part of all biological reactions rely on the action of cofactor-dependent enzymes. By adding the functionality of a cofactor to the protein matrix, enzymes have found a way of expanding their catalytic power that has led to an enormous collection of different biocatalysts. For the various types of cofactors observed in nature, different molecular approaches of cofactor binding and anchoring have been identified. Flavoproteins represent one of the major groups of cofactordependent enzymes (1, 2). However, data on the molecular process of FAD or FMN binding are scarce, while only a limited number of flavoprotein structures in the apo form are known (3-5). A striking example of the importance of efficient FAD binding was recently shown for methylenetetrahydrofolate reductase (6). A commonly found point mutation in the corresponding human gene results in a decreased cofactor affinity, which can lead to severe inborn health problems e.g. neuraltube defects and Down syndrome. For a better understanding of the process of flavin binding, we have started a study on the process of flavinylation in vanillyl-alcohol oxidase (VAO). 1 VAO (EC 1.1.3.38) is a FAD-dependent enzyme capable of oxidizing a wide range of phenolic substrates providing the fungus Penicillium simplicissimum with a tool for metabolizing aromatic compounds (7). The structure of VAO encompasses 560 residues and a fully buried covalently bound FAD cofactor ( Fig. 1) (8). The VAO monomer consists of two ␣/␤ domains: residues 1-270 and 500 -560 form a FAD binding domain, while the intervening residues form the so-called cap domain. The active site is located in the core of the protein, at the interface of the two domains with the flavin being covalently linked to His-422 of the cap domain. From a sequence alignment study it was recognized that VAO is a representative of a novel flavoprotein family, whose me...

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Crystal structure of apo‐glycolate oxidase

Cited by 14 publications

References 31 publications

Conformational Changes and Stabilization of Inosine 5′-Monophosphate Dehydrogenase Associated with Ligand Binding and Inhibition by Mycophenolic Acid

Conformational Changes and Stabilization of Inosine 5′-Monophosphate Dehydrogenase Associated with Ligand Binding and Inhibition by Mycophenolic Acid

Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

Structural Analysis of Flavinylation in Vanillyl-Alcohol Oxidase

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