TAILIEUCHUNG - Báo cáo khoa học: Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis

The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation (CCR) inBacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre). We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of ± lm for HPrSerP–CcpA and ± lmfor CrhP–CcpA complex dissoci-ation. | iFEBS Journal Quantitative interdependence of coeffectors CcpA and cre in carbon catabolite regulation of Bacillus subtilis Gerald Seidel Marco Diel Norbert Fuchsbauer and Wolfgang Hillen Lehrstuhl fur Mikrobiologie Institut fur Mikrobiologie Biochemie und Genetik der Friedrich-Alexander Universitat Erlangen-Nurnberg Germany Keywords carbon catabolite regulation CrhP HPrSerP fluorescence spectroscopy surface plasmon resonance Correspondence W. Hillen Lehrstuhl fur Mikrobiologie Institut fur Mikrobiologie Biochemie und Genetik der Friedrich-Alexander Universitat Erlangen-Nurnberg Staudtstr. 5 91058 Erlangen Germany Fax 49 9131 8528082 Tel 49 9131 8528081 E-mail whillen@ Received 28 January 2005 revised 16 March 2005 accepted 23 March 2005 doi The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation CCR in Bacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements cre . We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance SPR revealing small and similar equilibrium constants of pM for HPrSerP-CcpA and pM for CrhP-CcpA complex dissociation. Forty millimolar fructose-1 6-bisphosphate FBP or glucose-6-phos-phate Glc6-P increases the affinity of HPrSerP to CcpA at least twofold but have no effect on CrhP-CcpA binding. Saturation of binding of CcpA to cre as studied by fluorescence and SPR is dependent on 50 pM of HPrSerP or 200 pM CrhP. The rate constants of HPrSerP-CcpA-cre complex formation are ka 3 1 X 106 M-1-s-1 and kd X 10 3-s_1 resulting in a KD of nM. FBP and Glc6-P stimulate CcpA-HPrSerP but not CcpA-CrhP binding to cre. Maximal HPr-SerP-CcpA-cre complex formation in the presence of 10 mM FBP requires about 10-fold less HPrSerP. These data suggest a specific role for FBP and Glc6-P in enhancing only .

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