Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae

Author:

López Geovani1,Quezada Héctor1,Duhne Mariana1,González James1,Lezama Mijail1,El-Hafidi Mohammed2,Colón Maritrini1,Martínez de la Escalera Ximena1,Flores-Villegas Mirelle Citlali1,Scazzocchio Claudio34,DeLuna Alexander5,González Alicia1

Affiliation:

1. Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

2. Departamento de Biomedicina Cardiovascular, Instituto Nacional Cardiología Ignacio Chávez, Juan Badiano No. 1, Colonia Sección XVI, Tlalpan, Mexico City, Mexico

3. Deptartment of Microbiology, Imperial College London, South Kensington Campus, London, United Kingdom

4. Institut de Génétique et Microbiologie, CNRS UMR 8621, Université Paris-Sud, Orsay, France

5. Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y Estudios Avanzados del IPN, Irapuato, Guanajuato, Mexico

Abstract

ABSTRACT Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9 , that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4 Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4 Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo . Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine–α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

Publisher

American Society for Microbiology

Subject

Molecular Biology,General Medicine,Microbiology

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