Crystal Structures of Biotin Protein Ligase from Pyrococcus horikoshii OT3 and its Complexes: Structural Basis of Biotin Activation

Bagautdinov, B.; Kuroishi, Chizu; Sugahara, M.; Kunishima, N.

doi:10.1016/j.jmb.2005.08.032

Cited by 56 publications

(78 citation statements)

References 26 publications

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“…In the liganded structures, the loop becomes ordered and closes over the bound substrates to form the main hole of the active site. This ligand-induced ordering of the active site loop is also observed in the structures of wild-type PhBPL (9). The cocrystallization of PhBPL* with ATP and biotin provided the PhBPL*⅐biotinyl-5Ј-AMP complex where the U-shaped biotinyl-5Ј-AMP was found in the bifurcated main hole of BPL*, indicating the retained functionality of the single mutant (Fig.…”

Section: Resultssupporting

confidence: 60%

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Protein Biotinylation Visualized by a Complex Structure of Biotin Protein Ligase with a Substrate

Bagautdinov

Matsuura

Bagautdinova

et al. 2008

Journal of Biological Chemistry

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Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL⅐BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5-AMP, thereby providing the complex crystals. The four crystallographically independent BPL⅐BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction.

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Section: Resultssupporting

confidence: 60%

“…Overall, all the structures were found isomorphous to the wild-type PhBPL (9 53 is disordered. In the liganded structures, the loop becomes ordered and closes over the bound substrates to form the main hole of the active site.…”

Section: Resultsmentioning

confidence: 91%

Protein Biotinylation Visualized by a Complex Structure of Biotin Protein Ligase with a Substrate

Bagautdinov

Matsuura

Bagautdinova

et al. 2008

Journal of Biological Chemistry

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“…The most plausible common model suggests an involvement of Lys-142 to promote nucleophilic attack of the -amino group from the external lipoyl domain, which ultimately leads to a stable amide-like octanoyl-protein product. This hypothesis is supported by recent structural data from a complex of BirA from Pyrococcus horikoshii complexes with biotinyl-5Ј-AMP (28). In this complex, the equivalent invariant lysine is within hydrogenbonding distance of one of the carboxylate oxygen atoms in the biotin moiety, thereby increasing the polarization of the reactive group to allow -amino amide bond formation with the biotinyl acceptor domain.…”

Section: Resultsmentioning

confidence: 63%

The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase

Zhao

Eddine

et al. 2006

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

104

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Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked activesite complex with decanoic acid. We provide evidence that the transferase functions as a cysteine͞lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid͞base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.catalytic dyad ͉ lipoic acid ͉ x-ray structure ͉ thioester formation ͉ mass spectrometry S everal multicomponent enzyme complexes that catalyze key metabolic reactions in the citric acid cycle and single-carbon metabolism are posttranslationally modified by attachment to lipoic acid (1). These systems share a domain that covalently binds lipoic acid by means of an amide bond to the -amino group of a conserved exposed lysine residue. In many organisms, lipoylation is catalyzed by two separate enzymes, lipoyl protein ligase A (LplA) or octanoyl-[acyl carrier protein]-protein transferase (LipB; see Fig. 5, which is published as supporting information on the PNAS web site). Although LplA uses exogenous lipoic acid, LipB transfers endogenous octanoic acid, which is attached by means of a thioester bond to the 4Ј-phosphopantetheine cofactor of acyl carrier protein (ACP) onto lipoyl domains (2-4). These octanoylated domains are converted into lipoylated derivatives by the S-adenosyl-L-methioninedependent enzyme, lipoyl synthase (LipA), which catalyzes the insertion of sulfur atoms into the six-and eight-carbon positions of the corresponding fatty acid (5-7). This process bypasses the requirement for an exogenous supply of lipoic acid.In bacteria, enzymes involved in lipoylation have gained increasing attention because of their implication in pathogenicity. For instance, a Listeria monocytogenes mutant strain lacking LplA has been found to be defective in its ability to grow in the host cytosol and is less virulent in animals because of its dependence on host-derived lipoic acid (8). Mice Lias Ϫ/Ϫ (LipA) null variants die during embryogenesis, indicating that the mammalian pathway is related to the bacterial LipB͞LipA pathway and is essenti...

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“…For example, Pyrococcus horikoshii encodes a monofunctional ligase, which does not contain an N-terminal DNA binding domain and therefore does not bind to DNA. High-resolution structures of this ligase reveal significant structural homology between it and the C-terminal and central domains of BirA (35). Nonetheless, two striking differences are observed.…”

Section: Discussionmentioning

confidence: 98%

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

Naganathan

Beckett

2007

Journal of Molecular Biology

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The Escherichia coli biotin repressor, BirA, is an allosteric transcription regulatory protein to which binding of the small ligand corepressor, biotinyl-5'-AMP, promotes homodimerization and subsequent DNA binding. Structural data indicate that the apo-or unliganded repressor is characterized by four partially disordered loops that are ordered in the ligand-bound dimer. While three of these loops participate directly in the dimerization, the fourth, consisting of residues 212-234 is distal to the interface. This loop, which is ordered around the adenine ring of the adenylate in the BirA adenylate structure, is referred to as the adenylate binding loop or ABL. Although residues in the loop do not directly interact with the ligand, a hydrophobic cluster consisting of a tryptophan and two valine side chains assembles over the adenine base. Results of previous measurements suggest that folding of the ABL is integral to the allosteric response. This idea and the role of the hydrophobic cluster in the process were investigated by systematic replacement of each side chain in the cluster with alanine and analysis of the variant proteins for small ligand binding and dimerization. Isothermal titration calorimetry measurements indicate defects in adenylate binding for all ABL variants. Additionally, sedimentation equilibrium measurements reveal that coupling between adenylate binding and dimerization is compromised in each mutant. Partial proteolysis measurements indicate that the mutants are defective in ligand-linked folding of the ABL. These results indicate that the hydrophobic cluster is critical to the ligand-induced disorder-to-order transition in the ABL and that this transition is integral to the allosteric response in the biotin repressor.

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Crystal Structures of Biotin Protein Ligase from Pyrococcus horikoshii OT3 and its Complexes: Structural Basis of Biotin Activation

Cited by 56 publications

References 26 publications

Protein Biotinylation Visualized by a Complex Structure of Biotin Protein Ligase with a Substrate

Protein Biotinylation Visualized by a Complex Structure of Biotin Protein Ligase with a Substrate

The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

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