Molecular architecture of the Mn2+-dependent lactonase UlaG reveals an RNase-like metallo-beta-lactamase fold and a novel quaternary structure.
The ulaG gene, located in the ula regulon, is crucial for the catabolism of l-ascorbate under anaerobic conditions and it has been proposed to encode for the putative l-ascorbate-6-P lactonase. The ulaG gene is widespread among eubacteria, including human commensal and pathogenic genera such as Escherichia, Shigella, Klebsiella and Salmonella. Here, we report the three-dimensional structures of the apoenzyme and Mn(2+) holoenzyme of UlaG from E. coli to 2.6 A resolution, determined using single-wavelength anomalous diffraction phasing and molecular replacement, respectively. The structures reveal a highly specialized metallo-beta-lactamase-like fold derived from an ancient structural template that was involved in RNA maturation and DNA repair. This fold has a novel quaternary architecture consisting of a hexameric ring formed by a trimer of UlaG dimers. A mononuclear Mn(2)(+)-binding site resides at the core of the active site, which displays micromolar affinity for Mn(2+) and a distorted trigonal bipyramidal coordination. The active site Mn(2+) ion can be replaced by Co(2+) or Zn(2+), but not by Fe(3+). We further show that the Mn(2+) or Co(2)(+)-loaded enzyme exhibits lactonase activity towards l-ascorbate 6-P, thereby providing the first direct evidence of its catalytic role in the L-ascorbate catabolic pathway. Guided by the structural homology, we show that UlaG is able to cleave phosphodiester linkages in cyclic nucleotides, suggesting that the conservation of the fold and of the key catalytic residues allows for the evolutionary acquisition of substrate specificity for novel but related substrates.
Garces
Fernando
F
Department of Biochemistry and Molecular Biology, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, ES 08028, Spain.
Fernández
Francisco José
FJ
Montellà
Cristina
C
Penya-Soler
Esther
E
Prohens
Rafel
R
Aguilar
Juan
J
Baldomà
Laura
L
Coll
Miquel
M
Badia
Josefa
J
Vega
M Cristina
MC
D001205:Ascorbic Acid / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D003035:Cobalt / Q000378:metabolism; D018360:Crystallography, X-Ray; D020536:Enzyme Activators / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D007501:Iron / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry
D020536:Enzyme Activators; D003035:Cobalt; D008345:Manganese; D007501:Iron; D002265:Carboxylic Ester Hydrolases; C527649:L-ascorbate 6-phosphate lactonase, E coli; D001618:beta-Lactamases; D015032:Zinc; D001205:Ascorbic Acid