Functional analyses of a highly thermostable hexokinase from Pyrobaculum calidifontis. (January 2023)

• Record Type: Journal Article
• Authors: Shakir, Nisar Ahmed; Aslam, Mehwish; Bibi, Tahira; Falak, Samia; Rashid, Naeem
• Is Part Of: Carbohydrate research. Volume 523(2023)
• Publication Date: 2023-01
• Subjects: Hexokinase -- Thermostable -- Functional analysis -- Pyrobaculum calidifontis -- Crenarchaeota; Carbohydrates -- Periodicals; Chemistry, Organic -- Periodicals; Biochemistry -- Periodicals; Carbohydrates -- Periodicals; Chimie organique -- Périodiques; Glucides -- Périodiques; Biochemistry; Carbohydrates; Chemistry, Organic; Periodicals; Electronic journals; 507.78
• DOI: 10.1016/j.carres.2022.108711
• Languages: English
• ISSNs: 0008-6215

Abstract

Abstract: The gene encoding a repressor open reading frame sugar kinase (ROK) family protein from hyperthermophilic crenarchaeon Pyrobaculum calidifontis, Pcal-HK, was cloned and expressed in Escherichia coli . The recombinant protein was produced in soluble and highly active form. Purified Pcal-HK was highly thermostable and existed in a monomeric form in solution. The enzyme was specific to ATP as phosphoryl donor but showed broad specificity to phosphoryl acceptors. It catalyzed the phosphorylation of a number of hexoses, including glucose, glucosamine, N-acetyl glucosamine, fructose and mannose, at nearly the same rate and similar affinity. The enzyme was metal ion dependent exhibiting highest activity at 90–95 °C and pH 8.5. Mg 2+ was most effective metal ion, which could be partially replaced by Mn 2+, Ni 2+ or Zn 2+ . Kinetic parameters were determined at 90 °C and the enzyme showed almost similar catalytic efficiency ( k cat / K m ) towards the above mentioned hexoses. To the best of our knowledge, Pcal-HK is the most active thermostable ROK family hexokinase characterized to date which catalyzes the phosphorylation of various hexoses with nearly similar affinity. Graphical abstract: Image 1 Highlights: Recombinant Pcal-HK was highly thermostable exhibiting highest activity at 90–95 °C. Pcal-HK was specific to ATP as phosphoryl donor but showed broad specificity towards phosphoryl acceptors. Pcal-HK catalyzed the phosphorylation of a number of hexoses at nearly the same rate and similar affinity. Pcal-HK is the most active and thermostable ROK family hexokinase characterized to date.