Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum.
The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD(+)-dependent Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 A resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 A. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 A and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Delta(1)-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Delta(1)-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.
Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, USA.
Dhiraj
D
Srivastava
Jonathan P
JP
Schuermann
Tommi A
TA
White
Navasona
N
Krishnan
Nikhilesh
N
Sanyal
Greg L
GL
Hura
Anmin
A
Tan
Michael T
MT
Henzl
Donald F
DF
Becker
John J
JJ
Tanner
D020369:Bradyrhizobium / Q000201:enzymology*; D003460:Crystallization; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D011392:Proline / Q000378:metabolism
D005420:Flavoproteins; D011392:Proline