Crystal structure of Rv1220c, a SAM-dependentO-methyltransferase fromMycobacterium tuberculosis

Rv1220c fromMycobacterium tuberculosisis annotated as anO-methyltransferase (MtbOMT). Currently, no structural information is available for this protein. Here, the crystal structure ofMtbOMT refined to 2.0 Å resolution is described. The structure reveals the presence of a methyltransferase fold and shows clear electron density for one molecule ofS-adenosylmethionine (SAM), which was apparently bound by the protein during its production inEscherichia coli. Although the overall structure ofMtbOMT resembles the structures ofO-methyltransferases fromCornybacterium glutamicum,Coxiella burnettiandAlfa alfa, differences are observed in the residues that make up the active site. Notably, substitution of Asp by His164 seems to abrogate metal binding byMtbOMT. A putative catalytic His–Asp pair located in the vicinity of SAM is absolutely conserved inMtbOMT homologues from all species ofMycobacterium, suggesting a conserved function for this protein.

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Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21009675 ◽

2021 ◽

Vol 77 (11) ◽

Author(s):

Kohei Sasamoto ◽

Tomoki Himiyama ◽

Kunihiko Moriyoshi ◽

Takashi Ohmoto ◽

Koichi Uegaki ◽

...

Keyword(s):

Crystal Structure ◽

Cellulose Acetate ◽

Electron Density ◽

Active Site ◽

Catalytic Domain ◽

Crystal Packing ◽

Signal Sequence ◽

Industrial Applications ◽

Side Chain ◽

Active Site Conformation

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

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Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17015813 ◽

2017 ◽

Vol 73 (12) ◽

pp. 651-656 ◽

Cited By ~ 1

Author(s):

Taichi Mizobuchi ◽

Risako Nonaka ◽

Motoki Yoshimura ◽

Katsumasa Abe ◽

Shouji Takahashi ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Active Site ◽

Metal Binding ◽

Bivalve Mollusc ◽

Active Site Residues ◽

X Ray ◽

Aspartate Racemase ◽

Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

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A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.001213 ◽

2018 ◽

Vol 293 (21) ◽

pp. 7993-8008 ◽

Cited By ~ 8

Author(s):

Subrata Debnath ◽

Dalibor Kosek ◽

Harichandra D. Tagad ◽

Stewart R. Durell ◽

Daniel H. Appella ◽

...

Keyword(s):

Crystal Structure ◽

Metal Ions ◽

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Phosphatases ◽

Random Order ◽

Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

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A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser

Scientific Reports ◽

10.1038/s41598-019-52825-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Si Hoon Park ◽

Jaehyun Park ◽

Sang Jae Lee ◽

Woo Seok Yang ◽

Sehan Park ◽

...

Keyword(s):

Electron Density ◽

Active Site ◽

Free Electron ◽

Free Electron Laser ◽

Structural Information ◽

Bacteriophage T4 ◽

Vital Role ◽

Spectrometric Analysis ◽

X Ray ◽

High Resolution Structure

Abstract The hydroxymethylation of cytosine bases plays a vital role in the phage DNA protection system inside the host Escherichia coli. This modification is known to be catalyzed by the dCMP hydroxymethylase from bacteriophage T4 (T4dCH); structural information on the complexes with the substrate, dCMP and the co-factor, tetrahydrofolate is currently available. However, the detailed mechanism has not been understood clearly owing to a lack of structure in the complex with a reaction intermediate. We have applied the X-ray free electron laser (XFEL) technique to determine a high-resolution structure of a T4dCH D179N active site mutant. The XFEL structure was determined at room temperature and exhibited several unique features in comparison with previously determined structures. Unexpectedly, we observed a bulky electron density at the active site of the mutant that originated from the physiological host (i.e., E. coli). Mass-spectrometric analysis and a cautious interpretation of an electron density map indicated that it was a dTMP molecule. The bound dTMP mimicked the methylene intermediate from dCMP to 5′-hydroxymethy-dCMP, and a critical water molecule for the final hydroxylation was convincingly identified. Therefore, this study provides information that contributes to the understanding of hydroxymethylation.

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Crystal Structure of the Active Site Mutant Form of Soluble Fumarate Reductase, Osm1

Crystals ◽

10.3390/cryst9100504 ◽

2019 ◽

Vol 9 (10) ◽

pp. 504 ◽

Cited By ~ 1

Author(s):

Kim ◽

Kwon ◽

Jung ◽

Chun ◽

Ha ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Flavin Adenine Dinucleotide ◽

Structural Information ◽

Redox Balance ◽

Underlying Mechanism ◽

Fumarate Reductase ◽

Mutant Form ◽

Characteristic Features ◽

Fad Binding

Soluble fumarate reductase is essential for survival under anaerobic conditions. This enzyme can maintain the redox balance in the cell by catalyzing the reduction of fumarate to succinate. Although the overall reaction mechanism of soluble fumarate reductase in yeast, Osm1, has been proposed by a previous structural study, the details of the underlying mechanism are not completely elucidated. The present study provides the structural information regarding the active site mutant form of Osm1 (R326A), thus, revealing that R326A mutation does not affect the substrate binding. Structural alterations of the residues surrounding the active site, and the missing 2nd flavin adenine dinucleotide (FAD) in the previously defined 2nd FAD binding site, were observed as characteristic features of the Osm1 R326A crystal structure. Based on these findings, we provided a clue that can explain the loss of activity of Osm1 R326A.

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Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16007548 ◽

2016 ◽

Vol 72 (6) ◽

pp. 490-499

Author(s):

Mirja Krause ◽

Tiila-Riikka Kiema ◽

Peter Neubauer ◽

Rik K. Wierenga

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Directed Evolution ◽

Crystal Structures ◽

Active Site ◽

Triosephosphate Isomerase ◽

Point Mutations ◽

Van Der Waals Interactions ◽

Side Chain ◽

Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

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Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

Applied and Environmental Microbiology ◽

10.1128/aem.69.4.2349-2355.2003 ◽

2003 ◽

Vol 69 (4) ◽

pp. 2349-2355 ◽

Cited By ~ 18

Author(s):

Yuji Nagata ◽

Zbyněk Prokop ◽

Soňa Marvanová ◽

Jana Sýkorová ◽

Marta Monincová ◽

...

Keyword(s):

Crystal Structure ◽

Mycobacterium Tuberculosis ◽

Amino Acid ◽

Active Site ◽

Homology Model ◽

Protein Family ◽

Sphingomonas Paucimobilis ◽

Amino Acid Residues ◽

Haloalkane Dehalogenase

ABSTRACT The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.

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