scholarly journals Structural and kinetic studies of bifunctional and monofunctional proline catabolic enzymes

2011 ◽  
Author(s):  
◽  
Dhiraj Srivastava
Keyword(s):  
Crystal Structure ◽  
Molecular Mechanism ◽  
Kinetic Studies ◽  
Structural Basis ◽  
Proline Dehydrogenase ◽  
Catabolic Enzymes ◽  
Disease Condition ◽  
P5c Dehydrogenase

PutAs, Proline dehydrogenase and P5C dehydrogenase are involved in the oxidation of proline to glutamate. Mutations in proline dehydrogenase and P5C dehydrogenase cause a disease condition known as hyperprolinemia. In this work I have solved the crystal structure of bifunctional PutA and P5C dehydrogenase. The structure of P5C dehydrogenase sheds light on the structural basis of hyperprolinemia. I have also used kinetic and thermodynamic methods to understand the molecular mechanism of hyperprolinemia.

scholarly journals The Molecular and Structural Basis of O-methylation Reaction in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

2019 ◽  
Vol 20 (7) ◽  
pp. 1533 ◽  
Author(s):  
Yucheng Zhao ◽  
Nana Wang ◽  
Ziwei Sui ◽  
Chuanlong Huang ◽  
Zhixiong Zeng ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Gene Duplication ◽  
Caffeic Acid ◽  
Molecular Mechanism ◽  
Evolutionary Relationship ◽  
Substrate Preference ◽  
Structural Basis ◽  
Hydroxyl Groups ◽  
Single Mutation ◽  
Precise Molecular Mechanism

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.

scholarly journals Structural basis of recruitment of RBM39 to DCAF15 by a sulfonamide molecular glue E7820

2019 ◽  
Author(s):  
Xinlin Du ◽  
Oleg Volkov ◽  
Robert Czerwinski ◽  
HuiLing Tan ◽  
Carlos Huerta ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Kinetic Analysis ◽  
Mechanism Of Action ◽  
E3 Ligase ◽  
Kinetic Studies ◽  
Structural Basis

AbstractE7820 and indisulam are two examples of aryl sulfonamides that recruit RBM39 to Rbx-Cul4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. In order to understand their mechanism of action, we carried out kinetic analysis on the recruitment of RBM39 to DCAF15 and solved a crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39. E7820 packs in a shallow pocket on the surface of DCAF15 and the resulting modified interface binds RBM39 through the α1 helix of RRM2 domain. Our kinetic studies revealed that aryl sulfonamide and RBM39 bind to DCAF15 in a synergistic manner. The structural and kinetic studies confirm aryl sulfonamides as molecular glues in the recruitment of RBM39 and provide a framework for future efforts to utilize DCAF15 to degrade other protein of interests.

scholarly journals Crystal structure of the dopamine N-acetyltransferase–acetyl-CoA complex provides insights into the catalytic mechanism

2012 ◽  
Vol 446 (3) ◽  
pp. 395-404 ◽  
Author(s):  
Kuo-Chang Cheng ◽  
Jhen-Ni Liao ◽  
Ping-Chiang Lyu
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Complex Structure ◽  
Kinetic Studies ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Acetyl Coa ◽  
Acetyl Transfer

The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an AANAT. Until the present study, no insect Dat structure had been solved, and, consequently, the structural basis for its acetyl-transfer activity was not well understood. We report in the present paper the high-resolution crystal structure for a D. melanogaster Dat–AcCoA (acetyl-CoA) complex obtained using one-edge (selenium) single-wavelength anomalous diffraction. A binding study using isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu47, Ser182 and Ser186 were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism.

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

scholarly journals Structural basis of antigen recognition: crystal structure of duck egg lysozyme

2017 ◽  
Vol 73 (11) ◽  
pp. 910-920 ◽  
Author(s):  
David Brent Langley ◽  
Ben Crossett ◽  
Peter Schofield ◽  
Jenny Jackson ◽  
Mahdi Zeraati ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Binding Affinity ◽  
Anas Platyrhynchos ◽  
Antigen Recognition ◽  
Pekin Duck ◽  
Structural Basis ◽  
Primary Sequence ◽  
Duck Egg ◽  
Differential Affinity

Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, no structures of DEL have so far been reported, and the situation had been complicated by the presence of multiple isoforms and conflicting reports of primary sequence. Here, the structures of two DEL isoforms from the eggs of the commonly used Pekin duck (Anas platyrhynchos) are reported. Using structural analyses in combination with mass spectrometry, non-ambiguous DEL primary sequences are reported. Furthermore, the structures and sequences determined here enable rationalization of the binding affinity of DEL for well documented landmark anti-lysozyme antibodies.

scholarly journals Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease.

1994 ◽  
Vol 91 (8) ◽  
pp. 2915-2919 ◽  
Author(s):  
K. R. Acharya ◽  
R. Shapiro ◽  
S. C. Allen ◽  
J. F. Riordan ◽  
B. L. Vallee
Keyword(s):  
Crystal Structure ◽  
Functional Divergence ◽  
Structural Basis
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