scholarly journals Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

2021 ◽  
Vol 23 (1) ◽  
pp. 65
Author(s):  
Ilya Kolyadenko ◽  
Anastasia Scherbakova ◽  
Kirill Kovalev ◽  
Azat Gabdulkhakov ◽  
Svetlana Tishchenko
Keyword(s):  
Proton Transport ◽  
Catalytic Properties ◽  
Geometry Optimization ◽  
Reduction Process ◽  
Binding Pocket ◽  
Amino Acid Residues ◽  
Substrate Binding Pocket ◽  
Concomitant Reduction ◽  
Double Amino Acid ◽  
Optimal Ph

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.

scholarly journals Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity

2004 ◽  
Vol 378 (2) ◽  
pp. 687-692 ◽  
Author(s):  
Carrie TSOI ◽  
Mikael WIDERSTEN ◽  
Ralf MORGENSTERN ◽  
Stellan SWEDMARK
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Fold Increase ◽  
Binding Pocket ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Substrate Binding Pocket ◽  
Site Selectivity ◽  
Km Value ◽  
Exogenous Compounds

The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure–activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146→Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in Km value) but 54-fold less efficient in sulphating dopamine (8-fold increase in Km value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.

Significant improvement to the catalytic properties of aspartate aminotransferase: Role of hydrophobic and charged residues in the substrate binding pocket

Biochemistry ◽  
1994 ◽  
Vol 33 (1) ◽  
pp. 90-97 ◽  
Author(s):  
Eleonore Koehler ◽  
Mark Seville ◽  
Joachim Jaeger ◽  
Ian Fotheringham ◽  
Michael Hunter ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Catalytic Properties ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Charged Residues

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa

Performance of poly-o-phenylenediamine and its carbon dot composite electrode in the removal of Cu2+ from its aqueous solution

2021 ◽  
pp. 2151037
Author(s):  
Yu Meng ◽  
Qing Zhong ◽  
Arzugul Muslim
Keyword(s):  
Aqueous Solution ◽  
Electrochemical Reduction ◽  
Composite Electrode ◽  
Reduction Process ◽  
Order Kinetic ◽  
Carbon Dot ◽  
First Order ◽  
Oxidation Reduction ◽  
Order Kinetic Model ◽  
Optimal Ph

Because −NH2 and −NH− in poly-[Formula: see text]-phenylenediamine (P[Formula: see text]PD) can interact strongly with the empty orbitals of Cu to show unique electrochemical activity, P[Formula: see text]PD is suitable for the removal of Cu[Formula: see text] by electrochemical oxidation–reduction process. In this study, with P[Formula: see text]PD and its carbon dot composite (CDs/P[Formula: see text]PD) as working electrodes, the electrochemical reduction and removal of Cu[Formula: see text] in the aqueous solution were carried out with the potentiostatic method. According to effects of voltage, pH of the solution, initial concentration of Cu[Formula: see text], and electrochemical reduction time on the Cu[Formula: see text] removal, the Cu[Formula: see text] removal ratios of P[Formula: see text]PD and CDs/P[Formula: see text]PD were up to 64.69% and 73.34%, respectively, at −0.2 V and the optimal pH. Additionally, results showed that these processes were in line with the quasi-first order kinetic model. Both P[Formula: see text]PD and CDs/P[Formula: see text]PD showed good reproducibility in six cycles. After five times of repeated usage, the regeneration efficiencies of P[Formula: see text]PD and CDs/P[Formula: see text]PD dropped to 77.04% and 79.36%, respectively.

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