Adenylyl cyclase activity of Cya1 from the cyanobacterium Synechocystis sp. strain PCC 6803 is inhibited by bicarbonate

Abstract

Bicarbonate stimulates the activities of several class III adenylyl cyclases studied to date. However, we show here that bicarbonate decreased V(max) and substrate affinity in Cya1, a major adenylyl cyclase in the cyanobacterium Synechocystis sp. strain PCC 6803. This indicates that manifestation of the bicarbonate responsiveness is specifically modulated in Cya1.

FIG. 1.

Adenylyl cyclase activity of Cya1 is inhibited by bicarbonate. (A) Activity was measured in the presence of 50 mM NaHCO₃, NaCl, and/or KCl as indicated. (B) Activity was assayed in the presence of various concentrations of NaHCO₃. The reaction mixture included 200 μM ATP.

FIG. 2.

Effects of bicarbonate on kinetic properties of Cya1 adenylyl cyclase activity. Activity was assayed as a function of substrate ATP concentrations in the absence (closed circles) or presence (open circles) of 50 mM NaHCO₃.

FIG. 3.

Amino acid sequence alignment of the catalytic region of Cya1 with various class III adenylyl cyclases. An amino acid sequence of Synechocystis Cya1 was obtained from the KAZUSA DNA Research Institute site at http://www.kazusa.or.jp/en/. Accession numbers for other aligned amino acid sequences are as follows: Anabaena CyaB1, BAA13998; Spirulina CyaC, BAA22997; Rattus sAC, AAD04035; Mycobacterium Rv1319c, Q10632; Mycobacterium Rv1264, Z77137; Rattus transmembrane AC (tmAC), M55075; and Mus tmAC9, CAA03415. Amino acids involved in substrate recognition (Lys-177), metal ion coordination (Asp-181), and transition state stabilization (Asn-258 and Arg-262) are indicated in bold type. As indicated in the right margin, the AC activities of Anabaena CyaB1, Spirulina CyaC, Rattus sAC, and Mycobacterium Rv1319c are stimulated by bicarbonate; however, those of Mycobacterium Rv1264 and Rattus tmAC are insensitive to bicarbonate (1, 2, 6). Bicarbonate has been proposed to mimic the carboxyl group of Asp conserved in the bicarbonate-insensitive ACs at the position of Thr-251 (italic type) (1). Alignment is based on the alignment of a previous report (1). Gaps introduced to maximize alignment are indicated by dashes.

FIG. 4.

Kinetic properties of adenylyl cyclase activity of N-terminal truncated Cya1. The activity of the purified N-terminal truncated version of Cya1 was assayed as a function of substrate ATP concentration in the absence (closed circles) or presence (open circles) of 50 mM NaHCO₃. Points reflecting substrate inhibition were omitted for regression analysis.