Biomedical subjects

Heidi L Schubert

Publications and source records attributed to Heidi L Schubert.

13 recordsLinked to original sources

PubMed2006

Structure of ATP-bound human ATP:cobalamin adenosyltransferase.

Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B12) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 A crystal structure of ATP bound to hATR refined to an Rfree value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

Adenosine Triphosphate↗

PubMed2005

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing.

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.

Adenosine Deaminase↗

PubMed2005

Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis.

Gun4 has been implicated in a developmental signaling pathway between the chloroplast and the nucleus involving magnesium protoporphyrin IX (MgP(IX)), the first dedicated intermediate in the chlorophyll biosynthetic pathway. Here we present the crystal structure of Thermosynechococcus elongatus Gun4 at 1.5 A, describe the binding affinities of Gun4 for substrate and product porphyrin molecules, and identify a likely (Mg)P(IX) binding site on the protein. Kinetic analyses show that Gun4 dramatically increases the efficiency of transformation of porphyrin substrate to metalloporphyrin product and that it also reduces the threshold Mg2+ concentration required for activity at low porphyrin concentration. Gun4 therefore controls magnesium chelatase at physiologically significant Mg2+ concentrations and likely acts as a molecular switch in vivo so that in its absence magnesium chelatase is inactive. This mechanism could allow Gun4 to mediate magnesium protoporphyrin levels both for chlorophyll biosynthesis and for signaling to the nucleus.

Amino Acid Sequence↗

PubMed2005

Ubiquitin-binding domains.

Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These recently discovered motifs interpret and transmit information conferred by protein ubiquitylation to control various cellular events. Detailed molecular structures are known for a number of UBDs, but to understand their mechanism of action, we also need to know how binding specificity is determined, how ubiquitin binding is regulated, and the function of UBDs in the context of full-length proteins. Such knowledge will be key to our understanding of how ubiquitin regulates cellular proteins and processes.

Carrier Proteins↗

PubMed2005

Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence.

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis.

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.

Amino Acid Sequence↗

PubMed2004

Ubiquitin recognition by the human TSG101 protein.

The UEV domain of the TSG101 protein functions in both HIV-1 budding and the vacuolar protein sorting (VPS) pathway, where it binds ubiquitylated proteins as they are sorted into vesicles that bud into late endosomal compartments called multivesicular bodies (MVBs). TSG101 UEV-ubiquitin interactions are therefore important for delivery of both substrates and hydrolytic enzymes to lysosomes, which receive proteins via fusion with MVBs. Here, we report the crystal structure of the TSG101 UEV domain in complex with ubiquitin at 2.0 A resolution. TSG101 UEV contacts the Ile44 surface and an adjacent loop of ubiquitin through a highly solvated interface. Mutations that disrupt the interface inhibit MVB sorting, and the structure also explains how the TSG101 UEV can independently bind its ubiquitin and Pro-Thr/Ser-Ala-Pro peptide ligands. Remarkably, comparison with mapping data from other UEV and related E2 proteins indicates that although the different E2/UEV domains share the same structure and have conserved ubiquitin binding activity, they bind through very different interfaces.

Alanine↗

PubMed2003

Structures along the catalytic pathway of PrmC/HemK, an N5-glutamine AdoMet-dependent methyltransferase.

Posttranslational methylation of release factors on the glutamine residue of a conserved GGQ motif is required for efficient termination of protein synthesis. This methylation is performed by an N(5)-glutamine methyltransferase called PrmC/HemK, whose crystal structure we report here at 2.2 A resolution. The electron density at the active site appears to contain a mixture of the substrates, S-adenosyl-L-methionine (AdoMet) and glutamine, and the products, S-adenosyl-L-homocysteine (AdoHcy) and N(5)-methylglutamine. The C-terminal domain of PrmC adopts the canonical AdoMet-dependent methyltransferase fold and shares structural similarity with the nucleotide N-methyltransferases in the active site, including use of a conserved (D/N)PPY motif to select and position the glutamine substrate. Residues of the PrmC (197)NPPY(200) motif form hydrogen bonds that position the planar Gln side chain such that the lone-pair electrons on the nitrogen nucleophile are oriented toward the methyl group of AdoMet. In the product complex, the methyl group remains pointing toward the sulfur, consistent with either an sp(3)-hybridized, positively charged Gln nitrogen, or a neutral sp(2)-hybridized nitrogen in a strained conformation. Due to steric overlap within the active site, proton loss and formation of the neutral planar methylamide product are likely to occur during or after product release. These structures, therefore, represent intermediates along the catalytic pathway of PrmC and show how the (D/N)PPY motif can be used to select a wide variety substrates.

Amino Acid Motifs↗

PubMed2003

A story of chelatase evolution: identification and characterization of a small 13-15-kDa "ancestral" cobaltochelatase (CbiXS) in the archaea.

The cobaltochelatase required for the synthesis of vitamin B12 (cobalamin) in the archaeal kingdom has been identified as CbiX through similarity searching with the CbiX from Bacillus megaterium. However, the CbiX proteins in the archaea are much shorter than the CbiX proteins found in eubacteria, typically containing less than half the number of amino acids in their primary structure. For this reason the shorter CbiX proteins have been termed CbiXS and the longer versions CbiXL. The CbiXS proteins from Methanosarcina barkeri and Methanobacter thermoautotrophicum were overproduced in Escherichia coli as recombinant proteins and characterized. Through complementation studies of a defined chelatase-deficient strain of E. coli and by direct in vitro assays the function of CbiXS as a sirohydrochlorin cobaltochelatase has been demonstrated. On the basis of sequence alignments and conserved active site residues we suggest that CbiXS may represent a primordial chelatase, giving rise to larger chelatases such as CbiXL, SirB, CbiK, and HemH through gene duplication and subsequent variation and selection. A classification scheme for chelatases is proposed.

Amino Acid Sequence↗

PubMed2003

Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.

Amino Acid Sequence↗

PubMed2003

Many paths to methyltransfer: a chronicle of convergence.

S-adenosyl-L-methionine (AdoMet) dependent methyltransferases (MTases) are involved in biosynthesis, signal transduction, protein repair, chromatin regulation and gene silencing. Five different structural folds (I-V) have been described that bind AdoMet and catalyze methyltransfer to diverse substrates, although the great majority of known MTases have the Class I fold. Even within a particular MTase class the amino-acid sequence similarity can be as low as 10%. Thus, the structural and catalytic requirements for methyltransfer from AdoMet appear to be remarkably flexible.

Animals↗

PubMed2002

The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase.

Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a beta-NAD(+)-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 A resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes.

Amino Acid Sequence↗

PubMed2002

The biosynthesis of adenosylcobalamin (vitamin B12).

Vitamin B12, or cobalamin, is one of the most structurally complex small molecules made in Nature. Major progress has been made over the past decade in understanding how this synthesis is accomplished. This review covers some of the most important findings that have been made and provides the reader with a complete description of the transformation of uroporphyrinogen III into adenosylcobalamin (AdoCbl). 183 references are cited.

Bacteria↗