Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 å resolution

doi:10.1016/S0969-2126(00)00042-3

Structure

Volume 2, Issue 5, May 1994, Pages 407-414

https://doi.org/10.1016/S0969-2126(00)00042-3 Get rights and content

Abstract

Background In Escherichia coli, the enzymes of the biotin biosynthesis pathway are encoded by the bio operon. One of these enzymes, ATP-dependent dethiobiotin synthetase, catalyzes the carboxylation of 7,8-diaminopelargonic acid leading to the formation of the ureido ring of biotin. The enzyme belongs to the class of ATP-dependent carboxylases and we present here the first crystal structure determined for this class of enzyme.

Results We have determined the crystal structure of homodimeric dethiobiotin synthetase to 1.65 å resolution. The subunit consists of a seven-stranded parallel β -sheet, surrounded by α -helices. The sheet contains the classical mononucleotide-binding motif with a fingerprint peptide Gly-X-X-X-X-X-Gly-Lys-Thr. The mononucleotide binding part of the structure is very similar to the GTP-binding protein H- ras -p21 and thus all GTP-binding proteins. A comparison reveals that some of the residues, which in H- ras -p21 interact with the nucleotide and the metal ion, are conserved in the synthetase.

Conclusions The three-dimensional structure of dethiobiotin synthetase has revealed that ATP-dependent carboxylases contain the classical mononucleotide-binding fold. Considerable similarities to the structure of the GTP-binding protein H- ras -p21 were found, indicating that both proteins might have evolved from a common ancestral mononucleotide-binding fold.

Introduction

Biotin (vitamin H) is a cofactor in many biological carboxylation reactions, but is present only in very small amounts in mammalian cells. The vitamin is synthesized in plants and some microorganisms. In Escherichia coli, the necessary enzymes of biotin biosynthesis are encoded in a gene cluster, the bio operon. This cluster consists of five genes, bioA, B, F, C and D [1]. The nucleotide sequence of the bio operon has been determined [2]. These genes encode five different enzymes, which are involved in the synthesis of biotin from pimeloyl-coenzyme A.

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of biotin, converting 7,8-diaminopelargonic acid to dethiobiotin (Figure 1). DTBS is a homodimer with a molecular weight of 24 kDa per subunit [3]. The enzyme is mechanistically of considerable interest, since it is an example of a carboxylase which does not use biotin as a coenzyme. Its mechanism is also different from the carboxylation reaction catalyzed by ribulose bisphosphate carboxylase in the fixation of CO ₂.

DTBS uses CO ₂ as second substrate and requires Mg-ATP as a cofactor. The proposed reaction pathway involves formation of a carbamate with one of the amines of the substrate which then reacts with Mg-ATP to give the mixed phosphoric-carbamic acid anhydride plus Mg-ADP. The mixed anhydride is then thought to react with the other amino group of the substrate to yield dethiobiotin and phosphate.

As the first step in a biochemical and structural characterization of the biotin biosynthesis pathway, we have crystallized DTBS from E. coli and determined its three- dimensional structure by protein crystallography. In this paper, we describe the refined three-dimensional structure of the enzyme which reveals the fold for an ATP-dependent carboxylase, at 1.65 å resolution.

Section snippets

Crystallization and structure determination

The crystals of DTBS are monoclinic, space group C2 with cell dimensions a = 73.2 å, b = 49.2 å and c = 61.8 å, β = 107.1° . A very similar crystal form has been reported recently using ammonium sulfate as precipitant [4]. The crystals are stable in the X-ray beam and diffract to very high resolution. On a conventional rotating anode, diffraction is observed to at least 1.65 å resolution. Based on a molecular weight of 24 kDa ([4], and Lorimer and Miller, unpublished data), the packing density

Biological implications

Biological carboxylation reactions are catalyzed by several classes of enzymes with different mechanisms. These classes include biotin-dependent enzymes, ribulose bisphosphate carboxylase, and the ATP-dependent carboxylases such as dethiobiotin synthetase (DTBS).

We have determined the crystal structure of DTBS. As this is the first structure of an ATP -dependent carboxylase, it reveals the fold of this class of enzyme. The enzyme is a homodimer, each monomer consisting of a seven-stranded

Acknowledgements

We thank Prof. Gerry Cohen, University of Tel Aviv for the gift of an overexpressing strain of E. coli. We also thank the following individuals for their contributions to the determination of the correct primary structure: Dr. Howard Hershey and Linda Besl for nucleotide sequencing, Tom Miller, Tom Webb and Pat Webber for amino acid sequencing and Dr. Barbara Larsen for electrospray MS.

Weijun Huang, Ylva Lindqvist and Gunter Schneider (corresponding author), Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center Box 590, S-751 24 Uppsala, Sweden.

Katharine J Gibson, Dennis Flint and George Lorimer, DuPont Central Research and Development, Experimental Station, PO Box 80402, Wilmington, DE 19880-0402, USA.

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Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase
2015, Tuberculosis
Citation Excerpt :
Without the ‘gate keeper’ asparagine, MtDTBS can accommodate a broad range of NTPs. It has previously been proposed that DTBS is more similar to GTP-binding proteins than ATP-dependent enzymes based on its fold [35] and the presence of the [NQ]-[KR]-x-[DE] GTP specificity motif in the E. coli, H. pylori and F. tularensis enzymes (containing NDVTP, NLKGN, and NCNDS motifs respectively) [18]. The first asparagine in this motif (numbering Asn175 in EcDTBS) was suggested to be an evolutionary relic from a protein able to bind both adenosine and guanine nucleotides and provides nucleotide specificity [18,34].
Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar K_m and k_cat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue.
Structural insights into HypB, a GTP-binding protein that regulates metal binding
2006, Journal of Biological Chemistry
Citation Excerpt :
There is another direct ionic interaction with Asp-75 from helix α3. The magnesium coordination sphere of HypB is complemented by Thr-47 from the P-loop, the β,γ-phosphate oxygens, and a water-mediated contact to the α-phosphate, very similar to what has been described for BioD (30). This is different from the coordination sphere of Ras and Ras-like proteins, which possess only two direct ionic interactions coming from two phosphate oxygens, two hydroxyl side chains, and two water molecules as ligands (Fig. 2C).
HypB is a prokaryotic metal-binding guanine nucleotide-binding protein that is essential for nickel incorporation into hydrogenases. Here we solved the x-ray structure of HypB from Methanocaldococcus jannaschii. It shows that the G-domain has a different topology than the Ras-like proteins and belongs to the SIMIBI (after Signal Recognition Particle, MinD and BioD) class of NTP-binding proteins. We show that HypB undergoes nucleotide-dependent dimerization, which is apparently a common feature of SIMIBI class G-proteins. The nucleotides are located in the dimer interface and are contacted by both subunits. The active site features residues from both subunits arguing that hydrolysis also requires dimerization. Two metal-binding sites are found, one of which is dependent on the state of bound nucleotide. A totally conserved ENV/IGNLV/ICP motif in switch II relays the nucleotide binding with the metal ionbinding site. The homology with NifH, the Fe protein subunit of nitrogenase, suggests a mechanistic model for the switch-dependent incorporation of a metal ion into hydrogenases.
Structural enzymology of biotin biosynthesis
2001, FEBS Letters
Over the last years, significant progress has been made in the understanding of the genetics and enzymology of the biosynthetic pathway of the vitamin biotin. The enzymes catalyzing the last four steps of this pathway, from pimeloyl-CoA to biotin, provide an ensemble of intriguing reaction mechanisms, which are presently being unravelled. The three-dimensional structures for three of these enzymes are known and provide a framework to which on-going mechanistic studies can be related.
Crystal structure of the bacterial cell division regulator MinD
2001, FEBS Letters
In bacterial cell division MinD plays a pivotal role, selecting the mid-cell over other sites. With MinC, MinD forms a non-specific inhibitor of division, that interacts with FtsZ. Specificity is provided by MinD’s interaction with MinE at the mid-cell. We have solved the crystal structure of MinD-1 from Archaeoglobus fulgidus to 2.6 Å by multiple anomalous dispersion. MinD is a classic nucleotide binding protein, related to nitrogenase iron proteins, which have a fold of a seven-stranded parallel β-sheet, surrounded by α-helices. Although MinD, unlike the proteins it interacts with and those it is structurally related to, is a monomer, not a dimer.
Biosynthesis of biotin and lipoic acid
2001, Vitamins and Hormones
The genetics and mechanistic enzymology of biotin biosynthesis have been the subject of much investigation in the last decade, owing to the interest for biotin production by fermentation, on the one hand, and for the design of inhibitors with potential herbicidal properties, on the other hand. Four enzymes are involved in the synthesis of biotin from its two precursors, alanine and pimeloyl-CoA. They are now well-characterized and the X-ray structures of the first three have been published.
8-Amino-7-oxopelargonic acid synthase is a pyridoxal 5′-phosphate (PLP) enzyme, very similar to other acyl-CoA α-oxoamine synthases, and its detailed mechanism has been determined. The origin of its specific substrate, pimeloyl-CoA, however, is not completely established. It could be produced by a modified fatty acid pathway involving a malonyl thioester as the starter. 7,8-Diaminopelargonic acid (DAPA) aminotransferase, although sharing sequence and folding homologies with other transaminases, is unique as it uses S-adenosylmethionine (AdoMet) as the NH₂ donor. The mechanism of dethiobiotin synthethase is also now well understood. It catalyzes the formation of the ureido ring via a DAPA carbamate activated with ATP
On the other hand, the mechanism of the last enzyme, biotin synthase, which has long raised a very puzzling problem, is only starting to be unraveled and appears indeed to be very complex. Biotin synthase belongs to the family of AdoMet-dependent enzymes that reductively cleave AdoMet into a deoxyadenosyl radical, and it is responsible for the homolytic cleavage of CH bonds. A first radical formed on dethiobiotin is trapped by the sulfur donor, which was found to be the iron-sulfur (FeS) center contained in the enzyme, and cyclization follows in a second step. Two important features come from these results: (1) a new role for an FeS center has been revealed, and (2) biotin synthase is not only a catalyst but also a substrate for the reaction.
Lipoate synthase, which catalyzes the formation of two CS bonds from octanoic acid, has a very high sequence similarity with biotin synthase. Although no in vitro enzymology has been carried out with lipoate synthase, the sequence homology as well as the results of in vivo studies support the conclusion that both enzymes are strongly mechanistically related.
The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP-SRP receptor complex
2000, Structure
Background: Protein targeting to the endoplasmic reticulum in eukaryotes and to the cell membrane in prokaryotes is mediated by the signal recognition particle (SRP) and its receptor (SR). Both contain conserved GTPase domains in the signal-peptide-binding proteins (SRP54 and Ffh) and the SR proteins (SRα and FtsY). These GTPases are involved in the regulation of protein targeting. Most studies so far have focussed on the SRP machinery of mammals and bacteria, leaving the SRP system of archaea less well understood.
Results: We report the crystal structure of the conserved GTPase (NG-Ffh) from the thermophilic archaeon Acidianus ambivalens at 2.0 Å resolution and of the Thr112→Ala mutant, which is inactive in GTP hydrolysis. This is the first structure of an SRP component from an archaeon and allows for a detailed comparison with related structures from Escherichia coli and thermophilic bacteria. In particular, differences in the conserved consensus regions for nucleotide binding and the subdomain interfaces are observed, which provide information about the regulation of the GTPase. These interactions allow us to propose a common signalling mechanism for the SRP–SR system.
Conclusions: The overall structure of SRP-GTPases is well conserved between bacteria and archaea, which indicates strong similarities in the regulation of the SRP-targeting pathway. Surprisingly, structure comparisons identified a homodimeric ATP-binding protein as the closest relative. A heterodimer model for the SRP–SR interaction is presented.

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Katharine J Gibson, Dennis Flint and George Lorimer, DuPont Central Research and Development, Experimental Station, PO Box 80402, Wilmington, DE 19880-0402, USA.

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Structure

Abstract

Introduction

Section snippets

Crystallization and structure determination

Biological implications

Acknowledgements

References (23)

The purification and properties of dethiobiotin synthetase

J. Biol. Chem

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin synthesis

J. Mol. Biol

Solvent content in proteins

J. Mol. Biol

Binding of nucleotides by proteins

Curr. Opin. Struct. Biol

The refined structure of the complex between adenylate kinase from beef heart mitochondrial matrix and its substrate AMP at 1.85 å resolution

J. Mol. Biol

Crystal structure of adenylosuccinate synthetase from Escherichia coli

J. Biol. Chem

Multiwire area X-ray diffractometers

Methods Enzymol

Regulation of the biotin operon in E. coli

Ann. N.Y. Acad. Sci

The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon

J. Biol. Chem

Between objectivity and subjectivity

Nature

Improved methods for building protein models in electron density maps and the location of errors in these models

Acta Crystallogr. A

Cited by (40)

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

Structural insights into HypB, a GTP-binding protein that regulates metal binding

Structural enzymology of biotin biosynthesis

Crystal structure of the bacterial cell division regulator MinD

Biosynthesis of biotin and lipoic acid

The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP-SRP receptor complex

Structure

Research ArticleCrystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 å resolution

Abstract

Introduction

Section snippets

Crystallization and structure determination

Biological implications

Acknowledgements

J. Biol. Chem

J. Mol. Biol

J. Mol. Biol

Curr. Opin. Struct. Biol

J. Mol. Biol

J. Biol. Chem

Methods Enzymol

Regulation of the biotin operon in E. coli

Ann. N.Y. Acad. Sci

The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon

J. Biol. Chem

Between objectivity and subjectivity

Nature

Improved methods for building protein models in electron density maps and the location of errors in these models

Acta Crystallogr. A

Research Article
Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 å resolution