scholarly journals Crystal structure of chorismate mutase fromBurkholderia phymatum

2018 ◽  
Vol 74 (4) ◽  
pp. 187-192 ◽  
Author(s):  
Oluwatoyin A. Asojo ◽  
Sandhya Subramanian ◽  
Jan Abendroth ◽  
Ilyssa Exley ◽  
Donald D. Lorimer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Structural Genomics ◽  
Active Site ◽  
Chorismate Mutase ◽  
Biological Warfare ◽  
Beneficial Bacteria ◽  
Sequence Identity ◽  
Resolution Structure

The bacteriumBurkholderia phymatumis a promiscuous symbiotic nitrogen-fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. OtherBurkholderiaspecies are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase fromB. phymatumwas produced and crystallized, and a 1.95 Å resolution structure is reported. This enzyme shares less than 33% sequence identity with other homologs of known structure. There are two classes of chorismate mutase: AroQ and AroH. The bacterial subclass AroQγ has reported roles in virulence. Chorismate mutase fromB. phymatumhas the prototypical AroQγ topology and retains the characteristic chorismate mutase active site. This suggests that substrate-based chorismate mutase inhibitors will not be specific and are likely to affect beneficial bacteria such asB. phymatum.

scholarly journals Crystal structure of chorismate mutase from Burkholderia thailandensis

2018 ◽  
Vol 74 (5) ◽  
pp. 294-299
Author(s):  
Oluwatoyin A. Asojo ◽  
David M. Dranow ◽  
Dmitry Serbzhinskiy ◽  
Sandhya Subramanian ◽  
Bart Staker ◽  
...  
Keyword(s):  
Chorismate Mutase ◽  
Biological Warfare ◽  
Monoclinic Space Group ◽  
Asymmetric Unit ◽  
Antibiotic Resistant ◽  
Burkholderia Thailandensis ◽  
Sequence Identity ◽  
High Resolution Structure ◽  
Binding Cavity ◽  
Resolution Structure

Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic-resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease. The high-resolution structure of chorismate mutase from B. thailandensis was determined in the monoclinic space group P21 with three homodimers per asymmetric unit. The overall structure of each protomer has the prototypical AroQγ topology and shares conserved binding-cavity residues with other chorismate mutases, including those with which it has no appreciable sequence identity.

scholarly journals Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate

2002 ◽  
Vol 364 (1) ◽  
pp. 165-171 ◽  
Author(s):  
Peter H. REHSE ◽  
Ming ZHOU ◽  
Sheng-Xiang LIN
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bond ◽  
Active Site ◽  
Substrate Binding ◽  
Substrate Inhibition ◽  
Molecular Replacement ◽  
Alternative Substrate ◽  
Hydrophobic Residues ◽  
Sequence Identity ◽  
Binding Orientations

Dehydroepiandrosterone sulphotransferase (DHEA-ST) is an enzyme that converts dehydroepiandrosterone (DHEA), and some other steroids, into their sulphonated forms. The enzyme catalyses the sulphonation of DHEA on the 3α-oxygen, with 3′-phosphoadenosine-5′-phosphosulphate contributing the sulphate. The structure of human DHEA-ST in complex with its preferred substrate DHEA has been solved here to 1.99Å using molecular replacement with oestradiol sulphotransferase (37% sequence identity) as a model. Two alternative substrate-binding orientations have been identified. The primary, catalytic, orientation has the DHEA 3α-oxygen and the highly conserved catalytic histidine in nearly identical positions as are seen for the related oestradiol sulphotransferase. The substrate, however, shows rotations of up to 30°, and there is a corresponding rearrangement of the protein loops contributing to the active site. This may also reflect the low identity between the two enzymes. The second orientation penetrates further into the active site and can form a potential hydrogen bond with the desulphonated cofactor 3′,5′-phosphoadenosine (PAP). This second site contains more van der Waal interactions with hydrophobic residues than the catalytic site and may also reflect the substrate-inhibition site. The PAP position was obtained from the previously solved structure of DHEA-ST co-crystallized with PAP. This latter structure, due to the arrangement of loops within the active site and monomer interactions, cannot bind substrate. The results presented here describe details of substrate binding to DHEA-ST and the potential relationship to substrate inhibition.

High-resolution crystal structure of copper amine oxidase fromArthrobacter globiformis: assignment of bound diatomic molecules as O2

2013 ◽  
Vol 69 (12) ◽  
pp. 2483-2494 ◽  
Author(s):  
Takeshi Murakawa ◽  
Hideyuki Hayashi ◽  
Tomoko Sunami ◽  
Kazuo Kurihara ◽  
Taro Tamada ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Weight ◽  
Active Site ◽  
Amine Oxidase ◽  
Diatomic Molecules ◽  
Average Molecular Weight ◽  
Copper Amine Oxidase ◽  
Dimer Interface ◽  
Resolution Structure ◽  
Copper Amine

The crystal structure of a copper amine oxidase fromArthrobacter globiformiswas determined at 1.08 Å resolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight ∼200) as a cryoprotectant. The final crystallographicRfactor andRfreewere 13.0 and 15.0%, respectively. Several molecules of LMW PEG were found to occupy cavities in the protein interior, including the active site, which resulted in a marked reduction in the overallBfactor and consequently led to a subatomic resolution structure for a relatively large protein with a monomer molecular weight of ∼70 000. About 40% of the presumed H atoms were observed as clear electron densities in theFo−Fcdifference map. Multiple minor conformers were also identified for many residues. Anisotropic displacement fluctuations were evaluated in the active site, which contains a post-translationally derived quinone cofactor and a Cu atom. Furthermore, diatomic molecules, most likely to be molecular oxygen, are bound to the protein, one of which is located in a region that had previously been proposed as an entry route for the dioxygen substrate from the central cavity of the dimer interface to the active site.

scholarly journals Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

2020 ◽  
Vol 295 (51) ◽  
pp. 17514-17534
Author(s):  
Jūrate˙ Fahrig-Kamarauskait≑ ◽  
Kathrin Würth-Roderer ◽  
Helen V. Thorbjørnsrud ◽  
Susanne Mailand ◽  
Ute Krengel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
High Activity ◽  
Active Site ◽  
Shikimate Pathway ◽  
Catalytic Efficiency ◽  
Chorismate Mutase ◽  
Full Potential ◽  
Evolutionary Trajectories ◽  
Essential Enzyme

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

scholarly journals High-resolution structure of an atypical α-phosphoglucomutase related to eukaryotic phosphomannomutases

2013 ◽  
Vol 69 (10) ◽  
pp. 2008-2016 ◽  
Author(s):  
Przemyslaw Nogly ◽  
Pedro M. Matias ◽  
Matteo de Rosa ◽  
Rute Castro ◽  
Helena Santos ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Haloacid Dehalogenase ◽  
Enzyme Active Site ◽  
High Resolution Structure ◽  
Dimeric Form ◽  
Strict Specificity ◽  
Resolution Structure

The first structure of a bacterial α-phosphoglucomutase with an overall fold similar to eukaryotic phosphomannomutases is reported. Unlike most α-phosphoglucomutases within the α-D-phosphohexomutase superfamily, it belongs to subclass IIb of the haloacid dehalogenase superfamily (HADSF). It catalyzes the reversible conversion of α-glucose 1-phosphate to glucose 6-phosphate. The crystal structure of α-phosphoglucomutase fromLactococcus lactis(APGM) was determined at 1.5 Å resolution and contains a sulfate and a glycerol bound at the enzyme active site that partially mimic the substrate. A dimeric form of APGM is present in the crystal and in solution, an arrangement that may be functionally relevant. The catalytic mechanism of APGM and its strict specificity towards α-glucose 1-phosphate are discussed.

scholarly journals The Aspergillus fumigatus Sialidase Is a 3-Deoxy-d-glycero-d-galacto-2-nonulosonic Acid Hydrolase (KDNase)

2011 ◽  
Vol 286 (12) ◽  
pp. 10783-10792 ◽  
Author(s):  
Judith C. Telford ◽  
Juliana H. F. Yeung ◽  
Guogang Xu ◽  
Milton J. Kiefel ◽  
Andrew G. Watts ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Aspergillus Fumigatus ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Chair Conformation ◽  
Boat Conformation ◽  
Transition State Analog ◽  
Acetylneuraminic Acid ◽  
Nonulosonic Acid ◽  
Resolution Structure

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.

scholarly journals Structural elucidation of the heterodimeric cis-prenyltransferase NgBR/DHDDS complex reveals novel insights in regulation of protein glycosylation

2020 ◽  
Author(s):  
Ban Edani ◽  
Kariona A. Grabińska ◽  
Rong Zhang ◽  
Eon Joo Park ◽  
Benjamin Siciliano ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Protein Glycosylation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Chain Synthesis ◽  
Rate Limiting ◽  
Activity Comparison ◽  
Long Chain ◽  
Resolution Structure

SummaryCis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here we report the crystal structure of the human NgBR/DHDDS complex, which represents the first atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme’s active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme’s active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long chain polyprenol synthesis.

scholarly journals Structural elucidation of thecis-prenyltransferase NgBR/DHDDS complex reveals insights in regulation of protein glycosylation

2020 ◽  
Vol 117 (34) ◽  
pp. 20794-20802 ◽  
Author(s):  
Ban H. Edani ◽  
Kariona A. Grabińska ◽  
Rong Zhang ◽  
Eon Joo Park ◽  
Benjamin Siciliano ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Protein Glycosylation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Chain Synthesis ◽  
Rate Limiting ◽  
Activity Comparison ◽  
Long Chain ◽  
Resolution Structure

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimericcis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme’s active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulatecis-PTase activity. Comparison of NgBR/DHDDS with homodimericcis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme’s active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimericcis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.

scholarly journals Crystal structures of FolM alternative dihydrofolate reductase 1 from Brucella suis and Brucella canis

2022 ◽  
Vol 78 (1) ◽  
Author(s):  
Imani Porter ◽  
Trinity Neal ◽  
Zion Walker ◽  
Dylan Hayes ◽  
Kayla Fowler ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Structural Genomics ◽  
Structural Similarity ◽  
Biological Warfare ◽  
Infectious Agents ◽  
Sequence Identity ◽  
Brucella Canis ◽  
Brucella Suis ◽  
Dihydrofolate Reductases ◽  
Therapeutic Development

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported. The enzymes share ∼95% sequence identity but have less than 33% sequence identity to other homologues with known structure. The structures are prototypical NADPH-dependent short-chain reductases that share their highest tertiary-structural similarity with protozoan pteridine reductases, which are being investigated for rational therapeutic development.

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