Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture

2008 ◽  
Vol 413 (3) ◽  
pp. 417-427 ◽  
Author(s):  
Lynnette M. A. Dirk ◽  
Jack J. Schmidt ◽  
Yiying Cai ◽  
Jonathan C. Barnes ◽  
Katherine M. Hanger ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Gel Filtration ◽  
Peptide Deformylase ◽  
Ps Ii ◽  
D1 Polypeptide ◽  
Equivalent Residue ◽  
Essential Enzyme ◽  
Binding Subsites ◽  
Structural Determinant

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 Å (1 Å=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr178 as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr2 in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr178 to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr178 can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

Human Intestinal Maltase–Glucoamylase: Crystal Structure of the N-Terminal Catalytic Subunit and Basis of Inhibition and Substrate Specificity

2008 ◽  
Vol 375 (3) ◽  
pp. 782-792 ◽  
Author(s):  
Lyann Sim ◽  
Roberto Quezada-Calvillo ◽  
Erwin E. Sterchi ◽  
Buford L. Nichols ◽  
David R. Rose
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Catalytic Subunit ◽  
Human Intestinal Maltase

scholarly journals Crystal Structure of the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3B Provides Insights into Determinants of Substrate Specificity

2016 ◽  
Vol 291 (46) ◽  
pp. 24054-24064 ◽  
Author(s):  
Alexei Gorelik ◽  
Leonhard X. Heinz ◽  
Katalin Illes ◽  
Giulio Superti-Furga ◽  
Bhushan Nagar
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Acid Sphingomyelinase

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

2022 ◽  
Author(s):  
Jai Krishna Mahto ◽  
Neetu Neetu ◽  
Monica Sharma ◽  
Monika Dubey ◽  
Bhanu Prakash Vellanki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Polyethylene Terephthalate ◽  
Active Site ◽  
Catalytic Domain ◽  
Structural Features ◽  
Comamonas Testosteroni ◽  
Plastic Pollution ◽  
Microbial Utilization ◽  
Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

2017 ◽  
Vol 474 (20) ◽  
pp. 3373-3389 ◽  
Author(s):  
Dong-Dong Meng ◽  
Xi Liu ◽  
Sheng Dong ◽  
Ye-Fei Wang ◽  
Xiao-Qing Ma ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Complex Structure ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Substrate Selectivity ◽  
Glucose Residue ◽  
Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

scholarly journals The crystal structure of a xyloglucan-specific endo-β-1,4-glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity

FEBS Journal ◽  
2009 ◽  
Vol 276 (18) ◽  
pp. 5094-5100 ◽  
Author(s):  
Katsuro Yaoi ◽  
Hidemasa Kondo ◽  
Ayako Hiyoshi ◽  
Natsuko Noro ◽  
Hiroshi Sugimoto ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue

Crystal Structure of theβ-Subunit of Acyl-CoA Carboxylase:  Structure-Based Engineering of Substrate Specificity†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (44) ◽  
pp. 14027-14036 ◽  
Author(s):  
Lautaro Diacovich ◽  
Deborah Lynn Mitchell ◽  
Huy Pham ◽  
Gabriela Gago ◽  
Melrose Mendoza Melgar ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity

The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity

2015 ◽  
Vol 71 (3) ◽  
pp. 687-696 ◽  
Author(s):  
Utz Fischer ◽  
Simon Hertlein ◽  
Clemens Grimm
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Lipid A ◽  
Mechanistic Explanation ◽  
Binding Pocket ◽  
Multiple Drug ◽  
Structural Rearrangements ◽  
Multiple Drug Resistant ◽  
Pentose Sugar ◽  
Essential Enzyme

The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD+-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.

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