Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Tran, Kiet N; Jang, Sei-Heon; Lee, ChangWoo

Tran, Kiet N; Jang, Sei-Heon; Lee, ChangWoo.

Afiliación

Tran KN; Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University, Gyeongsan 38453, South Korea.
Jang SH; Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University, Gyeongsan 38453, South Korea.
Lee C; Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University, Gyeongsan 38453, South Korea. Electronic address: leec@daegu.ac.kr.

Biochim Biophys Acta Proteins Proteom ; 1869(1): 140543, 2021 01.

Article en En | MEDLINE | ID: mdl-32966894

RESUMEN

Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr177. In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.

Asunto(s)

Proteínas Bacterianas/química; Glucosa-6-Fosfato/química; Glucosafosfato Deshidrogenasa/química; Fenilalanina/química; Sphingomonas/química; Tirosina/química; Adaptación Fisiológica; Sustitución de Aminoácidos; Proteínas Bacterianas/genética; Proteínas Bacterianas/metabolismo; Sitios de Unión; Biocatálisis; Frío; Expresión Génica; Glucosa-6-Fosfato/metabolismo; Glucosafosfato Deshidrogenasa/genética; Glucosafosfato Deshidrogenasa/metabolismo; Enlace de Hidrógeno; Interacciones Hidrofóbicas e Hidrofílicas; Cinética; Simulación del Acoplamiento Molecular; Mutagénesis Sitio-Dirigida; Fenilalanina/metabolismo; Unión Proteica; Conformación Proteica en Hélice alfa; Conformación Proteica en Lámina beta; Dominios y Motivos de Interacción de Proteínas; Proteínas Recombinantes/química; Proteínas Recombinantes/genética; Proteínas Recombinantes/metabolismo; Alineación de Secuencia; Homología de Secuencia de Aminoácido; Sphingomonas/enzimología; Especificidad por Sustrato; Triptófano/química; Triptófano/metabolismo; Tirosina/metabolismo

Palabras clave

Active-site stability; Aromatic amino acids; Cold-adapted enzymes; Conformational flexibility; Glucose 6-phosphate dehydrogenase; Sphingomonas sp.

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Fenilalanina / Tirosina / Proteínas Bacterianas / Glucosa-6-Fosfato / Sphingomonas / Glucosafosfato Deshidrogenasa Idioma: En Revista: Biochim Biophys Acta Proteins Proteom Año: 2021 Tipo del documento: Article País de afiliación: Corea del Sur Pais de publicación: Países Bajos

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