Enzymatic properties of futalosine hydrolase, an enzyme essential to a newly identified menaquinone biosynthetic pathway.
In prokaryotes, menaquinone is used for respiration. In Escherichia coli, menaquinone is biosynthesized from chorismate by seven enzymes. However, very recently, we identified an alternative pathway (the futalosine pathway), which operates in some bacteria, including Streptomyces coelicolor, Helicobacter pylori, Campylobacter jejuni, and Thermus thermophilus. We describe the steps of this pathway, which branches at chorismate in a manner similar to the known pathway, but then follows a different route. This new pathway includes futalosine, an unusual nucleoside derivative consisting of inosine and o-substituted benzoate moieties, as a biosynthetic intermediate. In this study, a recombinant futalosine hydrolase (TTHA0556) of T. thermophilus, which participates in the second step of the pathway and catalyzes the reaction releasing hypoxanthine from futalosine, was prepared and used in functional analyses. Recombinant TTHA0556 formed a homotetramer and reacted only with futalosine; other structurally related nucleotides and nucleosides were not accepted. Recombinant TTHA0556 required no cofactors, and the optimum pH and temperature were 4.5 and 80 degrees C. The Km value was calculated to be 154.0+/-5.3 microM and the kcat value was 1.02/s. Recombinant TTHA0556 was slightly inhibited by hypoxanthine, with a Ki value of 1.1 mM.
Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan.
Tomoshige
T
Hiratsuka
Nobuya
N
Itoh
Haruo
H
Seto
Tohru
T
Dairi
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019271:Hypoxanthine / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009705:Nucleosides / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000378:metabolism; D024482:Vitamin K 2 / Q000378:metabolism*
D008670:Metals; D009705:Nucleosides; D011993:Recombinant Fusion Proteins; C122040:futalosine; D024482:Vitamin K 2; D019271:Hypoxanthine; D006867:Hydrolases