Comparison of two type IV hyperthermophilic adenylyl cyclases characterizations from the archaeon Pyrococcus furiosus

In this paper, two genes that encoded two soluble type IV adenylyl cyclases (AC) from the hyperthermophilic archaeon Pyrococcus furiosus (PFAC I and PFAC II) were cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Amino acid sequence analysis of the two enzymes showed 29% homology. PFAC I and PFAC II were both Mn2+ activated enzyme. They were purified by His-trap chromatography and had a specific activity of 3.1 × 103 U/mg at pH 10.0, 95 °C (PFAC I) and 2.0 × 103 U/mg at pH 11.0, 95 °C (PFAC II), respectively. The Km and kcat of PFAC I was 1.38 mM and 1.11 s−1. The Km and kcat of PFAC II was 1.44 mM and 0.80 s−1. The thermostability of PFAC I and PFAC II were higher than the soluble type IV adenylyl cyclases from Yersinia pestis (YpAC-IV). All of the properties suggested that these two adenylyl cyclases may be useful for the industrial producing of cyclic adenosine 3′,5′-monophosphate (cAMP).

cAMP from ATP by adenylate cyclase from Pyrococcus furiosus.Figure optionsDownload as PowerPoint slideHighlights
► Two type IV adenylyl cyclases from Pyrococcus furiosus have been expressed and characterized in this work.
► Both enzymes are Mg2+ activated and the optimal temperature of them is 95 °C.
► The specific activity of PFAC I and PFAC II are 3.1 × 103 U/mg and 2.0 × 103 U/mg.