Structural analysis of the sulfatase AmAS from Akkermansia muciniphila

2021 ◽  
Vol 77 (12) ◽  
Author(s):  
Chang-Cheng Li ◽  
Xin-Yue Tang ◽  
Yi-Bo Zhu ◽  
Ying-Jie Song ◽  
Ning-Lin Zhao ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Structural Characteristics ◽  
Substrate Binding ◽  
Degradation Process ◽  
Active Site Residues ◽  
Structural Variations ◽  
Akkermansia Muciniphila ◽  
Host Microbe Interactions

Akkermansia muciniphila, an anaerobic Gram-negative bacterium, is a major intestinal commensal bacterium that can modulate the host immune response. It colonizes the mucosal layer and produces nutrients for the gut mucosa and other commensal bacteria. It is believed that mucin desulfation is the rate-limiting step in the mucin-degradation process, and bacterial sulfatases that carry out mucin desulfation have been well studied. However, little is known about the structural characteristics of A. muciniphila sulfatases. Here, the crystal structure of the premature form of the A. muciniphila sulfatase AmAS was determined. Structural analysis combined with docking experiments defined the critical active-site residues that are responsible for catalysis. The loop regions I–V were proposed to be essential for substrate binding. Structure-based sequence alignment and structural superposition allow further elucidation of how different subclasses of formylglycine-dependent sulfatases (FGly sulfatases) adopt the same catalytic mechanism but exhibit diverse substrate specificities. These results advance the understanding of the substrate-recognition mechanisms of A. muciniphila FGly-type sulfatases. Structural variations around the active sites account for the different substrate-binding properties. These results will enhance the understanding of the roles of bacterial sulfatases in the metabolism of glycans and host–microbe interactions in the human gut environment.

scholarly journals The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 5053
Author(s):  
Alina K. Bakunova ◽  
Alena Yu. Nikolaeva ◽  
Tatiana V. Rakitina ◽  
Tatiana Y. Isaikina ◽  
Maria G. Khrenova ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Structural Analysis ◽  
Active Site ◽  
Active Sites ◽  
Structural Basis ◽  
Active Site Residues ◽  
Type Iv ◽  
Fold Type ◽  
Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

scholarly journals Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena

2020 ◽  
Vol 295 (17) ◽  
pp. 5751-5760
Author(s):  
Haehee Lee ◽  
Sangkee Rhee
Keyword(s):  
Binding Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Nitrogen Storage ◽  
Active Site Residues ◽  
Substrate Channeling ◽  
Arginine Dihydrolase ◽  
Arginine Catabolism ◽  
Cyanobacterium Anabaena ◽  
Ornithine Cyclodeaminase

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

scholarly journals Computational Approach Choice in Modeling Flexible Enzyme Active Sites

2019 ◽  
Author(s):  
Hisham Dokainish ◽  
James Gauld
Keyword(s):  
Conformational Changes ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Experimental Studies ◽  
Md Simulations ◽  
Acid Formation ◽  
Nucleophilic Attack ◽  
Sulfenic Acid ◽  
Proper Position

<div><div><div><p>The last step in the reductase step of the catalytic mechanism of MsrB was re-investigated using several computational approaches. Our previous QM-cluster paper showed that two possible mechanisms could occur, however the direct formation of disulfide from sulfonium cation was favored over sulfenic acid formation. In contrary, experimental studies suggest sulfenic acid formation. Therefore, first, we investigated the effect of level of theory, which confirmed previous conclusion. In addition, the effect of model choice was also investigated using ONIOM including a large QM layer around Cys440. Interestingly, deprotonating Cys440 leads to direct nucleophilic attack on Cys495 forming disulfide. Second, to eliminate the possibility that all previous results are an artifact of the used crystal structure in which the S...S distance is 3.29 Å, we ran a 5 ns MD simulation on the sulfonium cation intermediate. Surprisingly, our results show that the distance between the two sulfur is significantly increased to 4.88 Å. More importantly a water molecule is located in a proper position for nucleophilic attack. QM/MM calculations shows that sulfenic acid is formed via low barrier of 16.7 kJ mol-1. Finally, the effect of substrate binding on the two Cys's distance were investigated via running several MD simulations of possible intermediates, showing that substrate binding induces conformational changes increasing the sulfur's distance which is decreased upon substrate removal upon sulfenic acid formation. These results question the applicability of QM cluster approach in systems including flexible turns. It also emphasizes the importance of proper preparation of the starting structure.</p></div></div></div>

scholarly journals Computational Approach Choice in Modeling Flexible Enzyme Active Sites

2019 ◽  
Author(s):  
Hisham Dokainish ◽  
James Gauld
Keyword(s):  
Conformational Changes ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Experimental Studies ◽  
Md Simulations ◽  
Acid Formation ◽  
Nucleophilic Attack ◽  
Sulfenic Acid ◽  
Proper Position

<div><div><div><p>The last step in the reductase step of the catalytic mechanism of MsrB was re-investigated using several computational approaches. Our previous QM-cluster paper showed that two possible mechanisms could occur, however the direct formation of disulfide from sulfonium cation was favored over sulfenic acid formation. In contrary, experimental studies suggest sulfenic acid formation. Therefore, first, we investigated the effect of level of theory, which confirmed previous conclusion. In addition, the effect of model choice was also investigated using ONIOM including a large QM layer around Cys440. Interestingly, deprotonating Cys440 leads to direct nucleophilic attack on Cys495 forming disulfide. Second, to eliminate the possibility that all previous results are an artifact of the used crystal structure in which the S...S distance is 3.29 Å, we ran a 5 ns MD simulation on the sulfonium cation intermediate. Surprisingly, our results show that the distance between the two sulfur is significantly increased to 4.88 Å. More importantly a water molecule is located in a proper position for nucleophilic attack. QM/MM calculations shows that sulfenic acid is formed via low barrier of 16.7 kJ mol-1. Finally, the effect of substrate binding on the two Cys's distance were investigated via running several MD simulations of possible intermediates, showing that substrate binding induces conformational changes increasing the sulfur's distance which is decreased upon substrate removal upon sulfenic acid formation. These results question the applicability of QM cluster approach in systems including flexible turns. It also emphasizes the importance of proper preparation of the starting structure.</p></div></div></div>

Copper–oxygen Dynamics in Tyrosinase

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Sachiko Yanagisawa ◽  
Minoru Kubo ◽  
Genji Kurisu ◽  
Shinobu Itoh
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Active Form ◽  
Copper Ion ◽  
Atomic Resolution ◽  
Oxygen Species ◽  
X Ray ◽  
Oxygen Dynamics ◽  
Insight Into ◽  
Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu<sub>2</sub>O<sub>2</sub>core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.<br>

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa

2021 ◽  
Vol 77 (10) ◽  
pp. 1305-1316
Author(s):  
Yujing Chen ◽  
Haizhu Jia ◽  
Jianyu Zhang ◽  
Yakun Liang ◽  
Ruihua Liu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Class I ◽  
Polyamine Metabolism ◽  
Binding Assays ◽  
Starting Point ◽  
Family Activity ◽  
Living Organisms

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation–putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

scholarly journals Structure Characteristics, Biochemical Properties, and Pharmaceutical Applications of Alginate Lyases

Marine Drugs ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. 628
Author(s):  
Shu-Kun Gao ◽  
Rui Yin ◽  
Xiao-Chen Wang ◽  
Hui-Ning Jiang ◽  
Xiao-Xiao Liu ◽  
...  
Keyword(s):  
Brown Algae ◽  
Catalytic Mechanism ◽  
Degradation Products ◽  
Structural Characteristics ◽  
Catalytic Efficiency ◽  
Alginate Lyase ◽  
Biochemical Properties ◽  
Structure And Property ◽  
Alginate Oligosaccharides ◽  
Alginate Lyases

Alginate, the most abundant polysaccharides of brown algae, consists of various proportions of uronic acid epimers α-L-guluronic acid (G) and β-D-mannuronic acid (M). Alginate oligosaccharides (AOs), the degradation products of alginates, exhibit excellent bioactivities and a great potential for broad applications in pharmaceutical fields. Alginate lyases can degrade alginate to functional AOs with unsaturated bonds or monosaccharides, which can facilitate the biorefinery of brown algae. On account of the increasing applications of AOs and biorefinery of brown algae, there is a scientific need to explore the important aspects of alginate lyase, such as catalytic mechanism, structure, and property. This review covers fundamental aspects and recent developments in basic information, structural characteristics, the structure–substrate specificity or catalytic efficiency relationship, property, molecular modification, and applications. To meet the needs of biorefinery systems of a broad array of biochemical products, alginate lyases with special properties, such as salt-activated, wide pH adaptation range, and cold adaptation are outlined. Withal, various challenges in alginate lyase research are traced out, and future directions, specifically on the molecular biology part of alginate lyases, are delineated to further widen the horizon of these exceptional alginate lyases.

scholarly journals Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates.

2001 ◽  
Vol 48 (4) ◽  
pp. 995-1002 ◽  
Author(s):  
M Szabelski ◽  
K Stachowiak ◽  
W Wiczk
Keyword(s):  
Incubation Time ◽  
Active Sites ◽  
Initial Rate ◽  
Substrate Binding ◽  
Rapid Decrease ◽  
Fluorogenic Substrates ◽  
Binding Process ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Papain activity in a buffer containing Me2SO was studied using fluorogenic substrates. It was found that the number of active sites of papain decreases with increasing Me2SO concentration whereas the incubation time, in a buffer containing 3% Me2SO does not affect the number of active sites. However, an increase of papain incubation time in the buffer with 3% Me2SO decreased the initial rate of hydrolysis of Z-Phe-Arg-Amc as well as Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. Moreover, an increase of Me2SO concentration in working buffer decreased the initial rate of papain-catalysed hydrolysis of both substrates. A rapid decrease of the initial rate (by up to 30%) was observed between 1 and 2% Me2SO. Application of the Michaelis-Menten equation revealed that at the higher Me2SO concentrations the apparent values of k(cat)/Km decreased as a result of Km increase and kcat decrease. However, Me2SO changed the substrate binding process more effectively (Km) than the rate of catalysis k(cat).

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