Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

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Polyhydroxyalkanoates (PHA) production from synthetic waste using Pseudomonas pseudoflava : PHA synthase enzyme activity analysis from P. pseudoflava and P. palleronii

Bioresource Technology ◽

10.1016/j.biortech.2017.03.008 ◽

2017 ◽

Vol 234 ◽

pp. 99-105 ◽

Cited By ~ 22

Author(s):

M. Venkateswar Reddy ◽

Yasuteru Mawatari ◽

Rui Onodera ◽

Yuki Nakamura ◽

Yuka Yajima ◽

...

Keyword(s):

Enzyme Activity ◽

Activity Analysis ◽

Pha Synthase ◽

Pha Production

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The effect of pH on the kinetics of arylsulphatases A and B

Biochemical Journal ◽

10.1042/bj2130603 ◽

1983 ◽

Vol 213 (3) ◽

pp. 603-607 ◽

Cited By ~ 16

Author(s):

C O'Fagain ◽

B M Butler ◽

T J Mantle

Keyword(s):

Enzyme Activity ◽

Rat Liver ◽

Substrate Binding ◽

Acid Base ◽

Effect Of Ph ◽

Substrate Complex ◽

Enzyme Substrate ◽

General Acid ◽

Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

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Phospholipids modify substrate binding and enzyme activity of human cytochrome P450 27A1

The Journal of Lipid Research ◽

10.1194/jlr.m400300-jlr200 ◽

2004 ◽

Vol 45 (12) ◽

pp. 2345-2353 ◽

Cited By ~ 17

Author(s):

Dilyara A. Murtazina ◽

Ulla Andersson ◽

In-Su Hahn ◽

Ingemar Bjorkhem ◽

G. A. S. Ansari ◽

...

Keyword(s):

Enzyme Activity ◽

Cytochrome P450 ◽

Substrate Binding ◽

Human Cytochrome

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Combining free energy calculations with tailored enzyme activity assays to elucidate substrate binding of a phospho-lysine phosphatase

Chemical Science ◽

10.1039/d0sc03930f ◽

2020 ◽

Vol 11 (47) ◽

pp. 12655-12661

Author(s):

Anett Hauser ◽

Songhwan Hwang ◽

Han Sun ◽

Christian P. R. Hackenberger

Keyword(s):

Free Energy ◽

Enzyme Activity ◽

Molecular Docking ◽

Phosphatase Activity ◽

Substrate Binding ◽

Free Energy Calculations ◽

Energy Calculations

Combining phosphatase activity assays with molecular docking and free energy calculations reveals low promiscuity and substrate binding of intrinsically labile phospho-lysine peptides to the enzyme LHPP.

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Structural and functional analysis of Oceanobacillus iheyensis macrodomain reveals a network of waters involved in substrate binding and catalysis

Open Biology ◽

10.1098/rsob.160327 ◽

2017 ◽

Vol 7 (4) ◽

pp. 160327 ◽

Cited By ~ 6

Author(s):

Rubén Zapata-Pérez ◽

Fernando Gil-Ortiz ◽

Ana Belén Martínez-Moñino ◽

Antonio Ginés García-Saura ◽

Jordi Juanhuix ◽

...

Keyword(s):

Functional Analysis ◽

Enzyme Activity ◽

Substrate Binding ◽

Therapeutic Agents ◽

Water Molecules ◽

Substrate Conformation ◽

Coordinated Water ◽

Alkaliphilic Bacterium ◽

Hydrolysis Of

Macrodomains are ubiquitous conserved domains that bind or transform ADP-ribose (ADPr) metabolites. In humans, they are involved in transcription, X-chromosome inactivation, neurodegeneration and modulating PARP1 signalling, making them potential targets for therapeutic agents. Unfortunately, some aspects related to the substrate binding and catalysis of MacroD-like macrodomains still remain unclear, since mutation of the proposed catalytic aspartate does not completely abolish enzyme activity. Here, we present a functional and structural characterization of a macrodomain from the extremely halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiMacroD), related to hMacroD1/hMacroD2, shedding light on substrate binding and catalysis. The crystal structures of D40A, N30A and G37V mutants, and those with MES, ADPr and ADP bound, allowed us to identify five fixed water molecules that play a significant role in substrate binding. Closure of the β6–α4 loop is revealed as essential not only for pyrophosphate recognition, but also for distal ribose orientation. In addition, a novel structural role for residue D40 is identified. Furthermore, it is revealed that OiMacroD not only catalyses the hydrolysis of O -acetyl-ADP-ribose but also reverses protein mono-ADP-ribosylation. Finally, mutant G37V supports the participation of a substrate-coordinated water molecule in catalysis that helps to select the proper substrate conformation.

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Human placenta cytidine deaminase: proton-linked enzyme activity and substrate binding

IUBMB Life ◽

10.1080/15216549700202881 ◽

1997 ◽

Vol 42 (3) ◽

pp. 477-486

Author(s):

Silvia Vincenzetti ◽

Mauro Angeletti ◽

Giulio Lupidi ◽

Alessandra Cambi ◽

Paolo Natatini ◽

...

Keyword(s):

Enzyme Activity ◽

Human Placenta ◽

Cytidine Deaminase ◽

Substrate Binding

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Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

Biochemical Journal ◽

10.1042/bj20050263 ◽

2005 ◽

Vol 389 (1) ◽

pp. 173-180 ◽

Cited By ~ 19

Author(s):

Melinda DEMENDI ◽

Noboru ISHIYAMA ◽

Joseph S. LAM ◽

Albert M. BERGHUIS ◽

Carole CREUZENET

Keyword(s):

Enzyme Activity ◽

Substrate Specificity ◽

Molecular Basis ◽

Substrate Binding ◽

Structural Data ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Expected Effect ◽

Biochemical Assays ◽

Golden Opportunity

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

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