Crystal structures of UDP-N-acetylmuramic acid L-alanine ligase (MurC) from Mycobacterium bovis with and without UDP-N-acetylglucosamine

2021 ◽  
Vol 77 (5) ◽  
pp. 618-627
Author(s):  
Pil-Won Seo ◽  
Suk-Youl Park ◽  
Andreas Hofmann ◽  
Jeong-Sun Kim
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Mycobacterium Bovis ◽  
Domain Architecture ◽  
Binding Domain ◽  
Cross Linking ◽  
Nickel Ion ◽  
Binding Loop

Peptidoglycan comprises repeating units of N-acetylmuramic acid, N-acetylglucosamine and short cross-linking peptides. After the conversion of UDP-N-acetylglucosamine (UNAG) to UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, the crystal structure of Mycobacterium bovis MurC (MbMurC) is reported, which exhibits a three-domain architecture for the binding of UNAM, ATP and an amino acid as substrates, with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation that is not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of the three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety of the ligand, but not with the pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.

scholarly journals Transmembrane signaling in the sensor kinase DcuS ofEscherichia coli: A long-range piston-type displacement of transmembrane helix 2

2015 ◽  
Vol 112 (35) ◽  
pp. 11042-11047 ◽  
Author(s):  
Christian Monzel ◽  
Gottfried Unden
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Transmembrane Helix ◽  
Binding Domain ◽  
Cross Linking ◽  
Sensor Kinase ◽  
Amino Acid Residues ◽  
Terminal Part ◽  
Type Shift

The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane–water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21–41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181–201 in the OFF state and residues 185–205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2′ in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4–6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3–4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.

scholarly journals Anchoring the T6SS to the cell wall: Crystal structure of the peptidoglycan binding domain of the TagL accessory protein

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254232
Author(s):  
Van Son Nguyen ◽  
Silvia Spinelli ◽  
Éric Cascales ◽  
Alain Roussel ◽  
Christian Cambillau ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Protein Delivery ◽  
Cell Envelope ◽  
Interaction Network ◽  
Binding Domain ◽  
Target Cells ◽  
Accessory Protein ◽  
Type Vi Secretion System ◽  
Membrane Complex

The type VI secretion system (T6SS) is a widespread mechanism of protein delivery into target cells, present in more than a quarter of all sequenced Gram-negative bacteria. The T6SS constitutes an important virulence factor, as it is responsible for targeting effectors in both prokaryotic and eukaryotic cells. The T6SS comprises a tail structure tethered to the cell envelope via a trans-envelope complex. In most T6SS, the membrane complex is anchored to the cell wall by the TagL accessory protein. In this study, we report the first crystal structure of a peptidoglycan-binding domain of TagL. The fold is conserved with members of the OmpA/Pal/MotB family, and more importantly, the peptidoglycan binding site is conserved. This structure further exemplifies how proteins involved in anchoring to the cell wall for different cellular functions rely on an interaction network with peptidoglycan strictly conserved.

Structural studies of a cold-adapted dimeric β-D-galactosidase fromParacoccussp. 32d

2016 ◽  
Vol 72 (9) ◽  
pp. 1049-1061 ◽  
Author(s):  
Maria Rutkiewicz-Krotewicz ◽  
Agnieszka J. Pietrzyk-Brzezinska ◽  
Bartosz Sekula ◽  
Hubert Cieśliński ◽  
Anna Wierzbicka-Woś ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Domain Architecture ◽  
Glycoside Hydrolase Family ◽  
Molecular Replacement ◽  
Amino Acid Residues ◽  
Dimer Interface ◽  
Cold Adapted ◽  
Domain 3 ◽  
Hydrolysis Of

The crystal structure of a novel dimeric β-D-galactosidase fromParacoccussp. 32d (ParβDG) was solved in space groupP212121at a resolution of 2.4 Å by molecular replacement with multiple models using theBALBESsoftware. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric β-D-galactosidases fromEscherichia coliandArthrobactersp. C2-2, respectively. It is the second known structure of a cold-active GH2 β-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted β-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical β-D-galactosidase, the crystal structure in complex with galactose (ParβDG–Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric β-D-galactosidase reveals significant differences in comparison to other β-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 inParβDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

scholarly journals Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Misugi Uraji ◽  
Haruka Tamura ◽  
Eiichi Mizohata ◽  
Jiro Arima ◽  
Kun Wan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Ferulic Acid ◽  
Artificial Substrates ◽  
Feruloyl Esterase ◽  
Content Type ◽  
Agricultural Biomass ◽  
Catalytic Activities ◽  
Feruloyl Esterases

ABSTRACT Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus Streptomyces . Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high k cat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues. IMPORTANCE Streptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.

scholarly journals A Novel Two-domain Architecture Within the Amino Acid Kinase Enzyme Family Revealed by the Crystal Structure of Escherichia coli Glutamate 5-kinase

2007 ◽  
Vol 367 (5) ◽  
pp. 1431-1446 ◽  
Author(s):  
Clara Marco-Marín ◽  
Fernando Gil-Ortiz ◽  
Isabel Pérez-Arellano ◽  
Javier Cervera ◽  
Ignacio Fita ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Domain Architecture ◽  
Enzyme Family

scholarly journals Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition

2018 ◽  
Author(s):  
D. Sean Froese ◽  
Jola Kopec ◽  
Elzbieta Rembeza ◽  
Gustavo Arruda Bezerra ◽  
Anselm Erich Oberholzer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Methylenetetrahydrofolate Reductase ◽  
Binding Domain ◽  
Structural Basis ◽  
Methyl Donor ◽  
Tim Barrel ◽  
Conformational Plasticity ◽  
Methionine Cycle ◽  
Regulatory Connection

AbstractThe folate and methionine cycles are crucial to the biosynthesis of lipids, nucleotides and proteins, and production of the global methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region into proximity with the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16-total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation.

scholarly journals MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009276
Author(s):  
Xiaolong Liu ◽  
Jacob Biboy ◽  
Elisa Consoli ◽  
Waldemar Vollmer ◽  
Tanneke den Blaauwen
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Catalytic Activity ◽  
Cylindrical Part ◽  
Model System ◽  
Essential Proteins ◽  
Antibiotic Drug ◽  
Interaction Change ◽  
New Strategies

Rod-shape of most bacteria is maintained by the elongasome, which mediates the synthesis and insertion of peptidoglycan into the cylindrical part of the cell wall. The elongasome contains several essential proteins, such as RodA, PBP2, and the MreBCD proteins, but how its activities are regulated remains poorly understood. Using E. coli as a model system, we investigated the interactions between core elongasome proteins in vivo. Our results show that PBP2 and RodA form a complex mediated by their transmembrane and periplasmic parts and independent of their catalytic activity. MreC and MreD also interact directly with PBP2. MreC elicits a change in the interaction between PBP2 and RodA, which is suppressed by MreD. The cytoplasmic domain of PBP2 is required for this suppression. We hypothesize that the in vivo measured PBP2-RodA interaction change induced by MreC corresponds to the conformational change in PBP2 as observed in the MreC-PBP2 crystal structure, which was suggested to be the “on state” of PBP2. Our results indicate that the balance between MreC and MreD determines the activity of PBP2, which could open new strategies for antibiotic drug development.

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