Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase

Background: Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated. Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4- (2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1 /mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill’s body was suggested.

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Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.71.9.5290-5296.2005 ◽

2005 ◽

Vol 71 (9) ◽

pp. 5290-5296 ◽

Cited By ~ 11

Author(s):

Wei Zhang ◽

Yuan Liu ◽

Huabao Zheng ◽

Sheng Yang ◽

Weihong Jiang

Keyword(s):

Specific Activity ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Catalytic Function ◽

Alkaline Shift ◽

Key Residues

ABSTRACT In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k cat/Km ) compared to wild-type CA130. Their Km values were decreased by ca. 50%, and the k cat values increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

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Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

Biochemical Society Symposium ◽

10.1042/bss0710001 ◽

2004 ◽

Vol 71 ◽

pp. 1-14

Author(s):

David Leys ◽

Jaswir Basran ◽

François Talfournier ◽

Kamaldeep K. Chohan ◽

Andrew W. Munro ◽

...

Keyword(s):

Electron Transfer ◽

Dimensional Space ◽

Three Dimensional ◽

Kinetic Studies ◽

Molecular Assembly ◽

Conformational Sampling ◽

Trimethylamine Dehydrogenase ◽

Key Residues ◽

Electron Transferring Flavoprotein ◽

Electron Transfer Complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

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Faculty Opinions recommendation of MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020368.234435 ◽

2004 ◽

Author(s):

Michael Lenhard

Keyword(s):

Carotenoid Cleavage Dioxygenase ◽

Plant Signaling ◽

Signaling Molecule

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Faculty Opinions recommendation of Improving catalytic function by ProSAR-driven enzyme evolution.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1075795.528709 ◽

2007 ◽

Author(s):

Christopher Thanos

Keyword(s):

Enzyme Evolution ◽

Catalytic Function

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Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues.

Protein and Peptide Letters ◽

10.2174/0929866527666200613220245 ◽

2020 ◽

Vol 27 ◽

Author(s):

Sheetal Uppal ◽

Mohd. Asim Khan ◽

Suman Kundu

Keyword(s):

Molten Globule ◽

Life Forms ◽

Model System ◽

Heme Pocket ◽

The Past ◽

Specific Manner ◽

Delivery Vehicles ◽

The Stability ◽

Key Residues ◽

Synechocystis Sp

Aims: The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. Background: For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as “Synechocystis hemoglobin (SynHb)”. The “three histidines” linkages to heme are novel to this cyanobacterial hemoglobin. Objective: Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. Methods: Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. Result: The results revealed that the heme was stably associated to the protein under all denaturing conditions with His117 playing the anchoring role. The studies also highlighted the possibility of existence of a “molten globule” like intermediate at acidic pH in this exceptionally thermostable globin. His117 and other key residues in the heme pocket play an indispensable role in imparting significant polypeptide stability. Conclusion: Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.

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Computer-aided structural and molecular insights into the mechanisms by which Pseudouridimycin (PUM) disrupts cleft extension in bacterial RNA polymerase to block DNA entry and exit

Letters in Drug Design & Discovery ◽

10.2174/1570180817999201123165144 ◽

2020 ◽

Vol 17 ◽

Author(s):

Ali H. Rabbad ◽

Fisayo A. Olotu ◽

Mahmoud E. Soliman

Keyword(s):

Rna Polymerase ◽

Bacterial Infections ◽

Entry And Exit ◽

Dynamic Binding ◽

Binding Behavior ◽

Bacterial Rna ◽

Nucleotide Addition ◽

Switch Regions ◽

Cleft Extension ◽

Key Residues

Background: The ability of Pseudouridimycin (PUM) to occupy the nucleotide addition site of bacterial RNA Polymerase (RNAP) underlies its inhibitory potency as previously reported. PUM has gained high research interest as a broad-spectrum nucleoside analog that has demonstrated exciting potentials in treating drug-resistant bacterial infections. Objective: Herein, we identified, for the first time, a novel complementary mechanism by which PUM elicits its inhibitory effects on bacterial RNAP. Methods: The dynamic binding behavior of PUM to bacterial RNAP was studied using various dynamic analyses approaches. Results and Discussion: Findings revealed that in addition to occupying the nucleotide addition site, PUM also interrupts the unimpeded entry and exit of DNA by reducing the mechanistic extension of the RNAP cleft and perturbing the primary conformations of the switch regions. Moreover, PUM binding reduced the distances between key residues in the β and β’ subunits that extend to accommodate the DNA. Conclusion: This study’s findings present structural insights that would contribute to the structure-based design of potent and selective PUM inhibitors.

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Monotheism in Isaiah

The Oxford Handbook of Isaiah ◽

10.1093/oxfordhb/9780190669249.013.22 ◽

2020 ◽

pp. 218-248

Author(s):

Matthias Albani

Keyword(s):

Historical Context ◽

The Other ◽

Divine Power ◽

Crisis Situation ◽

Catalytic Function ◽

Other Hand ◽

The Future ◽

The One ◽

Religious Crisis

The monotheistic confession in Isa 40–48 is best understood against the historical context of Israel’s political and religious crisis situation in the final years of Neo-Babylonian rule. According to Deutero-Isaiah, Yhwh is unique and incomparable because he alone truly predicts the “future” (Isa 41:22–29)—currently the triumph of Cyrus—which will lead to Israel’s liberation from Babylonian captivity (Isa 45). This prediction is directed against the Babylonian deities’ claim to possess the power of destiny and the future, predominantly against Bel-Marduk, to whom both Nabonidus and his opponents appeal in their various political assertions regarding Cyrus. According to the Babylonian conviction, Bel-Marduk has the universal divine power, who, on the one hand, directs the course of the stars and thus determines the astral omens and, on the other hand, directs the course of history (cf. Cyrus Cylinder). As an antithesis, however, Deutero-Isaiah proclaims Yhwh as the sovereign divine creator and leader of the courses of the stars in heaven as well as the course of history on earth (Isa 45:12–13). Moreover, the conflict between Nabonidus and the Marduk priesthood over the question of the highest divine power (Sîn versus Marduk) may have had a kind of “catalytic” function in Deutero-Isaiah’s formulation of the monotheistic confession.

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A few key residues determine the high redox potential shift in azurin mutants

Organic & Biomolecular Chemistry ◽

10.1039/c5ob01819f ◽

2015 ◽

Vol 13 (45) ◽

pp. 11003-11013 ◽

Cited By ~ 20

Author(s):

Laura Zanetti-Polzi ◽

Carlo A. Bortolotti ◽

Isabella Daidone ◽

Massimiliano Aschi ◽

Andrea Amadei ◽

...

Keyword(s):

Redox Potential ◽

Potential Shift ◽

Key Residues ◽

High Redox Potential

The changes in the redox potential of Azurin upon mutation stem from the effects of a few key residues, including non-mutated ones, rather than being the result of a generalized rearrangement.

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