Point mutations in Candida glabrata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (CgHMGR) decrease enzymatic activity and substrate/inhibitor affinity

Abstract3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a crucial enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (CgHMGR) to explore the function of key amino acid residues in enzymatic activity. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A, and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was included. Regarding recombinant and wild-type CgHMGR, in vitro enzymatic activity was significantly lower for the former, as was the in silico binding energy of simvastatin, alpha-asarone and the HMG-CoA substrate. E711Q displayed the lowest enzymatic activity and binding energy, suggesting the importance of Glu711 (in the substrate binding site). The double mutant CgHMGR E680Q-M807R exhibited the second lowest enzymatic activity. Based on the values of the kinetic parameters KM and Vmax, the mutated amino acids appear to participate in binding. The current findings provide insights into the role of residues in the catalytic site of CgHMGR.

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Point Mutations In Candida Glabrata 3-Hydroxy-3-Methyl Glutaryl Coenzyme A Reductase (HMGRCg) An Antifungal Target, Decrease Enzymatic Activity And Substrate/Inhibitor Affinity

10.21203/rs.3.rs-618523/v1 ◽

2021 ◽

Author(s):

Dulce Andrade-Pavón ◽

Vanessa Fernández-Muñoz ◽

Wendy González-Ibarra ◽

César Hernández-Rodríguez ◽

J. Antonio Ibarra ◽

...

Keyword(s):

Binding Energy ◽

Enzymatic Activity ◽

Binding Site ◽

Candida Glabrata ◽

Coenzyme A ◽

Substrate Binding ◽

Point Mutations ◽

Substrate Binding Site ◽

Substrate Inhibitor ◽

Coenzyme A Reductase

Abstract An alternative target for antifungal drugs is 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR), a key enzyme in the ergosterol biosynthesis pathway. The aim of this study was to obtain, purify, characterize, and overexpress five point mutations in highly conserved regions of the catalytic domain of Candida glabrata HMGR (HMGRCg) to explore the function of key amino acid residues. Glutamic acid (Glu) was substituted by glutamine in the E680Q mutant (at the dimerization site), Glu by glutamine in E711Q (at the substrate binding site), aspartic acid by alanine in D805A and methionine by arginine in M807R (the latter two at the cofactor binding site). A double mutation, E680Q-M807R, was also made. The in vitro enzymatic activity decreased significantly in all recombinant (versus wild-type) HMGRCg, and the in silico binding energy for simvastatin, alpha-asarone and the substrate HMG-CoA was also lower for the mutants. The lowest enzymatic activity and binding energy was displayed by E711Q, suggesting that Glu711 (in the substrate binding site) is an important residue for enzymatic activity. The double mutant HMGRCg E680Q-M807R exhibited the second lowest enzymatic activity. The current findings provide insights into the role of residues in the catalytic site of HMGRCg.

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Faculty Opinions recommendation of Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: trapping propargyl alcohol with an alpha-70-substituted MoFe protein.

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

10.3410/f.1014842.194865 ◽

2003 ◽

Author(s):

Mark Nelson

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Propargyl Alcohol ◽

Substrate Binding Site ◽

Mofe Protein ◽

Femo Cofactor

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Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190613152030 ◽

2020 ◽

Vol 21 (2) ◽

pp. 117-130 ◽

Cited By ~ 1

Author(s):

Mohammad J. Hosen ◽

Mahmudul Hasan ◽

Sourav Chakraborty ◽

Ruhshan A. Abir ◽

Abdullah Zubaer ◽

...

Keyword(s):

Virtual Screening ◽

Binding Site ◽

Glucose Transporter ◽

Substrate Binding ◽

Mutation Screening ◽

Homology Model ◽

Connective Tissue Disorder ◽

Crystallographic Structure ◽

Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

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