scholarly journals Structural basis for the strict exclusion of proline from the N-glycosylation sequon

2021 ◽  
Author(s):  
Daisuke Kohda ◽  
Yuya Taguchi ◽  
Takahiro Yamasaki ◽  
Marie Ishikawa ◽  
Yuki Kawasaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Essential Role ◽  
Ring Structure ◽  
Structural Basis ◽  
Side Chain ◽  
Ramachandran Plot ◽  
Amino Acid Motif ◽  
External Loop ◽  
Backbone Dihedral Angle

Abstract Oligosaccharyltransferase (OST) catalyzes oligosaccharide transfer to the Asn residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where Pro is strictly excluded at position X. Considering the unique structural properties of proline, this exclusion may not be surprising, but the structural basis for the rejection of Pro residues should be explained explicitly. The crystal structure of an archaeal OST in a complex with a sequon-containing peptide and dolichol-phosphate was determined to a 2.7 Å resolution. The sequon part in the peptide forms two inter-chain hydrogen bonds with a conserved amino acid motif, TIXE. We confirmed the essential role of the TIXE motif and the adjacent regions by extensive alanine-scanning of the external loop 5. A Ramachandran plot revealed that the ring structure of the Pro side chain is incompatible with the φ backbone dihedral angle around -150° in the rigid sequon-TIXE structure.

scholarly journals The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yuya Taguchi ◽  
Takahiro Yamasaki ◽  
Marie Ishikawa ◽  
Yuki Kawasaki ◽  
Ryuji Yukimura ◽  
...  
Keyword(s):  
Essential Role ◽  
Ring Structure ◽  
Structural Basis ◽  
Side Chain ◽  
Ramachandran Plot ◽  
Amino Acid Motif ◽  
External Loop ◽  
Backbone Dihedral Angle ◽  
Insight Into

AbstractOligosaccharyltransferase (OST) catalyzes oligosaccharide transfer to the Asn residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where Pro is strictly excluded at position X. Considering the unique structural properties of proline, this exclusion may not be surprising, but the structural basis for the rejection of Pro residues should be explained explicitly. Here we determined the crystal structure of an archaeal OST in a complex with a sequon-containing peptide and dolichol-phosphate to a 2.7 Å resolution. The sequon part in the peptide forms two inter-chain hydrogen bonds with a conserved amino acid motif, TIXE. We confirmed the essential role of the TIXE motif and the adjacent regions by extensive alanine-scanning of the external loop 5. A Ramachandran plot revealed that the ring structure of the Pro side chain is incompatible with the ϕ backbone dihedral angle around −150° in the rigid sequon-TIXE structure. The present structure clearly provides the structural basis for the exclusion of Pro residues from the N-glycosylation sequon.

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

scholarly journals Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase

2021 ◽  
Vol 4 (4) ◽  
pp. e202000919
Author(s):  
Cheng-Han Ho ◽  
Yoshimasa Takizawa ◽  
Wataru Kobayashi ◽  
Yasuhiro Arimura ◽  
Hiroshi Kimura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Essential Role ◽  
Histone H3 ◽  
Underlying Mechanism ◽  
Structural Basis ◽  
Histone H4 ◽  
Nucleosomal Histone

SET8 is solely responsible for histone H4 lysine-20 (H4K20) monomethylation, which preferentially occurs in nucleosomal H4. However, the underlying mechanism by which SET8 specifically promotes the H4K20 monomethylation in the nucleosome has not been elucidated. Here, we report the cryo-EM structures of the human SET8–nucleosome complexes with histone H3 and the centromeric H3 variant, CENP-A. Surprisingly, we found that the overall cryo-EM structures of the SET8–nucleosome complexes are substantially different from the previous crystal structure models. In the complexes with H3 and CENP-A nucleosomes, SET8 specifically binds the nucleosomal acidic patch via an arginine anchor, composed of the Arg188 and Arg192 residues. Mutational analyses revealed that the interaction between the SET8 arginine anchor and the nucleosomal acidic patch plays an essential role in the H4K20 monomethylation activity. These results provide the groundwork for understanding the mechanism by which SET8 specifically accomplishes the H4K20 monomethylation in the nucleosome.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Crystal structure of fibrinogen-Aα peptide 1–23 (F8Y) bound to bovine thrombin explains why the mutation of Phe-8 to tyrosine strongly inhibits normal cleavage at Arg-16

1997 ◽  
Vol 326 (3) ◽  
pp. 815-822 ◽  
Author(s):  
Michael G. MALKOWSKI ◽  
Philip D. MARTIN ◽  
Susan T. LORD ◽  
Brian F. P. EDWARDS
Keyword(s):  
Crystal Structure ◽  
Structural Basis ◽  
Side Chain ◽  
Asymmetric Unit ◽  
Bovine Thrombin ◽  
R Factor ◽  
Native Peptide ◽  
Cell Dimensions ◽  
Scissile Bond ◽  
Unit Cell Dimensions

A peptide containing residues 1–50 of the Aα-chain of fibrinogen, expressed as a fusion peptide with β-galactosidase, is rapidly cleaved by thrombin at Arg-16, similarly to whole fibrinogen. When Phe-8, which is highly conserved, is replaced with tyrosine (F8Y), the cleavage is slowed drastically [Lord, Byrd, Hede, Wei and Colby (1990) J. Biol. Chem. 265, 838–843]. To examine the structural basis for this result, we have determined the crystal structure of bovine thrombin complexed with a synthetic peptide containing residues 1–23 of fibrinogen Aα and the F8Y mutation. The crystals are in space group P43212, with unit-cell dimensions of a = 88.3 Å (1 Å = 0.1 nm), c = 195.5 Å and two complexes in the asymmetric unit. The final R factor is 0.183 for 2σ data from 7.0 to 2.5 Å resolution. There is continuous density for the five residues in the P3, P2, P1, P1′ and P2′ positions of the peptide (Gly-14f to Pro-18f) at the active site of thrombin, and isolated but well-defined density for Tyr-8f at position P9 in the hydrophobic pocket of thrombin. The tyrosine residue is shifted relative to phenylalanine in the native peptide because the phenol side chain is larger and makes a novel, intrapeptide hydrogen bond with Gly-14f. Adjacent peptide residues cannot form the hydrogen bonds that stabilize the secondary structure of the native peptide. Consequently, the ‘reaction’ geometry at the scissile bond, eight residues from the mutation, is perturbed and the peptide is mostly uncleaved in the crystal structure.

scholarly journals Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase

1997 ◽  
Vol 328 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Luis MATA ◽  
Marta ERRA-PUJADA ◽  
Jean-Claude GRIPON ◽  
Michel-Yves MISTOU
Keyword(s):  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Aminopeptidase Activity ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Terminal Residue ◽  
Bleomycin Hydrolase ◽  
Eukaryotic Organisms

PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal α-carboxy group plays a key role in the strict aminopeptidase activity.

scholarly journals Conformational flexibility within the small domain of human serine racemase

2020 ◽  
Vol 76 (2) ◽  
pp. 65-73 ◽  
Author(s):  
Chloe R. Koulouris ◽  
Benjamin D. Bax ◽  
John R. Atack ◽  
S. Mark Roe
Keyword(s):  
Crystal Structure ◽  
Serine Racemase ◽  
Side Chain ◽  
Ramachandran Plot ◽  
Aspartate Receptor ◽  
Domain Movement ◽  
Small Domain ◽  
Small Molecule Modulators ◽  
Β Sheet ◽  
Helical Region

Serine racemase (SR) is a pyridoxal 5′-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,β-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78–155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded β-sheet of the small domain, including changes in the φ–ψ angles of all three residues in the C-terminal β-strand (residues 149–151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue β-strand (residues 78–83), but in the holoenzyme the β-strand is only four residues (78–81) and His82 has φ–ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target.

Single-component and two-component para -nitrophenol monooxygenases: structural basis for their catalytic difference

2021 ◽  
Author(s):  
Yuan Guo ◽  
De-Feng Li ◽  
Jianting Zheng ◽  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Crystal Structure ◽  
Rational Design ◽  
Single Component ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Two Component ◽  
Group D

para -Nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative degraders, whereas via ‘Benzenetriol (BT) pathway’ in Gram-positive ones. ‘HQ pathway’ is initiated by a single-component PNP 4-monooxygenase and ‘BT pathway’ by a two-component PNP 2-monooxygenase. Their rigio-selectivity intrigues us to investigate their catalytic difference through structural study. PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). However, the mechanisms are unknown. Here, PnpA1 was structurally determined to be a member of group D flavin-dependent monooxygenases with an acyl-CoA dehydrogenase fold. The crystal structure and site-directed mutagenesis underlined the direct involvement of Arg100 and His293 in catalysis. The bulky side chain of Val292 was proposed to push the substrate towards FAD, hence positioning the substrate properly. A variant N450A was found with improved activity for 4NC and 2C4NP, probably because of the reduced steric hindrance. PnpA1 shows obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be determined by Thr296 and loop 449–454. Above all, our study allows the structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regio-selectivity was proposed: the different PNP binding manner determines their choice of ortho - or para -hydroxylation on PNP. IMPORTANCE Single-component PNP monoxygenases hydroxylate PNP at 4-position while two-component ones at 2-position. However, their catalytic and structural differences remain elusive. The structure of single-component PNP 4-monooxygenase has previously been determined. In this study, to illustrate their catalytic difference, we resolved the crystal structure of, PnpA1, a typical two-component PNP 2-monooxygenase. The roles of several key amino acid residues in substrate binding and catalysis were revealed and a variant with improved activities towards 4NC and 2C4NP was obtained. Moreover, through comparing the two types of PNP monooxygenases, a hypothesis was proposed to account for their catalytic difference, which gives us a better understanding of these two similar reactions at molecular level. And these results will also be of further aid in enzyme rational design in bioremediation and biosynthesis.

scholarly journals Crystal Structure of Cefditoren Complexed with Streptococcus pneumoniae Penicillin-Binding Protein 2X: Structural Basis for Its High Antimicrobial Activity

2007 ◽  
Vol 51 (11) ◽  
pp. 3902-3907 ◽  
Author(s):  
Mototsugu Yamada ◽  
Takashi Watanabe ◽  
Takako Miyara ◽  
Nobuyoshi Baba ◽  
Jun Saito ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Streptococcus Pneumoniae ◽  
Active Form ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Penicillin Binding Protein ◽  
Level Of Activity ◽  
High Level ◽  
Tract Infections

ABSTRACT Cefditoren is the active form of cefditoren pivoxil, an oral cephalosporin antibiotic used for the treatment of respiratory tract infections and otitis media caused by bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, Klebsiella pneumoniae, and methicillin-susceptible strains of Staphylococcus aureus. β-Lactam antibiotics, including cefditoren, target penicillin-binding proteins (PBPs), which are membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. To envision the binding of cefditoren to PBPs, we determined the crystal structure of a trypsin-digested form of PBP 2X from S. pneumoniae strain R6 complexed with cefditoren. There are two PBP 2X molecules (designated molecules 1 and 2) per asymmetric unit. The structure reveals that the orientation of Trp374 in each molecule changes in a different way upon the formation of the complex, but each forms a hydrophobic pocket. The methylthiazole group of the C-3 side chain of cefditoren fits into this binding pocket, which consists of residues His394, Trp374, and Thr526 in molecule 1 and residues His394, Asp375, and Thr526 in molecule 2. The formation of the complex is also accompanied by an induced-fit conformational change of the enzyme in the pocket to which the C-7 side chain of cefditoren binds. These features likely play a role in the high level of activity of cefditoren against S. pneumoniae.

Structural basis of a novel activity of bacterial 6-pyruvoyltetrahydropterin synthase homologues distinct from mammalian 6-pyruvoyltetrahydropterin synthase activity

2014 ◽  
Vol 70 (5) ◽  
pp. 1212-1223 ◽  
Author(s):  
Kyung Hye Seo ◽  
Ningning Zhuang ◽  
Young Shik Park ◽  
Ki Hun Park ◽  
Kon Ho Lee
Keyword(s):  
Active Site ◽  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Wild Type ◽  
Specific Enzyme ◽  
Aromatic Residues

Escherichia coli6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterinin vitrocompared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterinin vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.5 Å resolution, respectively. The structures are highly conserved at the active site and the Zn2+binding site. However, comparison of the eCTPS structures with those of mammalian PTPS homologues revealed that two specific residues, Trp51 and Phe55, that are not found in mammalian PTPS keep the substrate bound by stacking it with their side chains. Replacement of these two residues by site-directed mutagenesis to the residues Met and Leu, which are only found in mammalian PTPS, converted eCTPS to the mammalian PTPS activity. These studies confirm that these two aromatic residues in eCTPS play an essential role in stabilizing the substrate and in the specific enzyme activity that differs from the original PTPS activity. These aromatic residues Trp51 and Phe55 are a key signature of bacterial PTPS enzymes that distinguish them from mammalian PTPS homologues.

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