Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues*.

Autor: Kumar Y; Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune, 411008, India., Singh RK; Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune, 411008, India.; Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, 400 076, India., Hazra AB; Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune, 411008, India.; Department of Biology, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune, 411008, India.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2021 Dec 10; Vol. 22 (24), pp. 3414-3424. Date of Electronic Publication: 2021 Sep 15.
DOI: 10.1002/cbic.202100211
Abstrakt: Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.
(© 2021 Wiley-VCH GmbH.)
Databáze: MEDLINE
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