title: Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase.
abstract: Nature recycles L-proline by converting it to L-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Delta1-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl beta-D-glucopyranoside and the growth of native crystals that diffracted to 2.3 A resolution at Advanced Light Source beamline 4.2.2. The space group is P2(1)2(1)2(1), with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 A. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.
authors.affiliation: Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
authors.forename: Tommi A
authors.initials: TA
authors.lastname: White
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D011394:Proline Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D011394:Proline Oxidase