Giuseppe Forlani1, Boguslaw Nocek2,3, Milosz Ruszkowski4. 1. Department of Life Science and Biotechnology, University of Ferrara, via L. Borsari 46, 44100, Ferrara, Italy. flg@unife.it. 2. The Bioscience Division, Argonne National Laboratory, Lemont, IL, 60439, USA. 3. AbbVie Inc., North Chicago, IL, USA. 4. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61704, Poznan, Poland.
Abstract
BACKGROUND: The enzyme that catalyzes the last step in proline synthesis, δ1-pyrroline-5-carboxylate reductase, showed in most cases a distinct preference in vitro for NADPH as the electron donor. METHODS AND RESULTS: A Zymomonas mobilis gene coding for a δ1-pyrroline-5-carboxylate reductase was cloned and heterologously expressed, and the recombinant protein was purified and characterized. The enzyme showed higher affinity to, and higher catalytic rate with NADH, with a specific activity of about 600 nkat (mg protein)-1. The molecular basis of this feature was investigated by analysis of the dinucleotide binding domain in silico. CONCLUSIONS: We postulate that the main determinants of coenzyme preference for P5C reductases are the length and the sequence of the motif A, whereas the overall sequence identity is insufficient to predict it a priori. Results are discussed in view of the obligately fermentative metabolism of this bacterium.
BACKGROUND: The enzyme that catalyzes the last step in proline synthesis, δ1-pyrroline-5-carboxylate reductase, showed in most cases a distinct preference in vitro for NADPH as the electron donor. METHODS AND RESULTS: A Zymomonas mobilis gene coding for a δ1-pyrroline-5-carboxylate reductase was cloned and heterologously expressed, and the recombinant protein was purified and characterized. The enzyme showed higher affinity to, and higher catalytic rate with NADH, with a specific activity of about 600 nkat (mg protein)-1. The molecular basis of this feature was investigated by analysis of the dinucleotide binding domain in silico. CONCLUSIONS: We postulate that the main determinants of coenzyme preference for P5C reductases are the length and the sequence of the motif A, whereas the overall sequence identity is insufficient to predict it a priori. Results are discussed in view of the obligately fermentative metabolism of this bacterium.
Authors: Eric F Pettersen; Thomas D Goddard; Conrad C Huang; Gregory S Couch; Daniel M Greenblatt; Elaine C Meng; Thomas E Ferrin Journal: J Comput Chem Date: 2004-10 Impact factor: 3.376
Authors: Kirsty Milne; Jianhui Sun; Esther A Zaal; Jenna Mowat; Patrick H N Celie; Alexander Fish; Celia R Berkers; Giuseppe Forlani; Fabricio Loayza-Puch; Craig Jamieson; Reuven Agami Journal: Bioorg Med Chem Lett Date: 2019-07-25 Impact factor: 2.823