title: The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.
abstract: Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.
authors: *Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av/Fuentenueva s/n, 18071 Granada, Spain.
authors: Noel
authors: N
authors: Mesa-Torres
authors: †Departmento de Cristalografia y Biología Estructural, Instituto de Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain.
authors: Cristina
authors: C
authors: Yunta
authors: ‡Center for Biomedical Research on Rare Diseases (CIBERER), University Hospital of the Canary Islands and CIBICAN, University of La Laguna, 38320 Tenerife, Spain.
authors: Israel
authors: I
authors: Fabelo-Rosa
authors: †Departmento de Cristalografia y Biología Estructural, Instituto de Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain.
authors: Juana María
authors: JM
authors: Gonzalez-Rubio
authors: *Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av/Fuentenueva s/n, 18071 Granada, Spain.
authors: José M
authors: JM
authors: Sánchez-Ruiz
authors: ‡Center for Biomedical Research on Rare Diseases (CIBERER), University Hospital of the Canary Islands and CIBICAN, University of La Laguna, 38320 Tenerife, Spain.
authors: Eduardo
authors: E
authors: Salido
authors: †Departmento de Cristalografia y Biología Estructural, Instituto de Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain.
authors: Armando
authors: A
authors: Albert
authors: *Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av/Fuentenueva s/n, 18071 Granada, Spain.
authors: Angel L
authors: AL
authors: Pey
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D003460:Crystallization; D018360:Crystallography, X-Ray; D056947:Enzyme Replacement Therapy / Q000379:methods*; D004795:Enzyme Stability; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000235:genetics / Q000628:therapy; D016415:Sequence Alignment; D012995:Solubility; D000637:Transaminases / Q000235:genetics / Q000627:therapeutic use*
chemical_list: D000637:Transaminases; C019635:Alanine-glyoxylate transaminase