Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

6A, NW12/2003G116

Crystallographic analysis of complex structures of xenobiotics-degrading enzyme
CumA1A2
Xue Song DONG1, Shinya FUSHINOBU*1, So-Young JUN1, Hideaki NOJIRI2, Hirofumi SHOUN1,Hisakazu YAMANE2, Toshio OMORI3, Takayoshi WAKAGI1
1
Dept. of Biotechnology, 2Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan
3
Dept. of Industrial Chemistry, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo
108-8548, Japan
Introduction
The cumene degradation pathway of Pseudomonas
fluorescens IP01 [1] begins with the hydroxylation of thearomatic ring by a cumene dioxygenase electron transportsystem (CumA) composed of three components. A flavincontaining ferredoxin reductase (CumA4) and a Rieske[2Fe-2S] ferredoxin (CumA3) transfer electrons from
NADH to the terminal oxygenase component(CumA1A2). Resultant diol compound is converted to acatechol derivative (3-isopropylchatecol) by a dihydrodiol
dehydrogenase (CumB). 3-Isopropylcatechol is attacked
by an extradiol dioxygenase (CumC), resulting in aformation of a meta-cleavage product (MCP), namely 2hydroxy-6-oxo-7-methylocta-2,4-dienoicacid(HOMODA). HOMODA is hydrolyzed by MCP
hydrolase (CumD), yielding isobutyric acid and 2hydroxypenta-2,4-dienoic acid (HODA). HODA is
eventually converted into the intermediates of the central
metabolism through the tricarboxylic acid cycle. We havealready determined the crystal structure of CumD S103A
mutant complexed with a cleavage product, isobutyricacid [2]. We conducted a comprehensive structure-based
study on the cumene degradation system.
Results and Discussion
CumA1A2
We solved the crystal structure of cumene dioxygenase(CumA1A2) by the molecular replacement method using
the structure of naphthalene dioxygenase (NDO; 1EG9)
as a search model. The structure is refined to R = 19.1%
and Rfree = 21.4% at 2.3 Å resolution [3]. The overallstructure was similar to NDO and composed of α3β3hexamer. The α subunit contains a Rieske [2Fe-2S]cluster and the active site with a non-heme mononuclear
ferrous ion. Main chain traces of the loops forming theactive site of CumA1A2 was largely deviated form thoseof NDO, and the shape of inner surface was also different.
CumA4
We succeeded in producing yellow crystals of CumA4.
Diffraction data was collected at BL-6A up to 2.3 Åresolution. The space group was C2221 and the cell
constants were, a = 79.1, b = 178.3, c = 236.8 Å. The

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Users' Reportstructure was solved by the molecular replacement
method using the structure of ferredoxin reductase
component of biphenyl dioxygenase (1F3P) as a searchmodel. An asymmetric unit contained three subunits. The
structure is currently under refinement.
CumCDiffraction data of a crystal of CumC was collected at
BL-18B up to 2.4 Å resolution. The structure was solvedby the molecular replacement method using the structureof 2,3-dihydroxybiphenyl 1,2-dioxygenase (1LGT) as asearch model. The structure is currently refined to R =25.6% and Rfree = 30.4%. However, one of the two CumC
molecules in the asymmetric unit appeared severely
disordered, and the electron density peak of the ferrous
ion at the active site appeared to be too weak. We aretrying to obtain more reliable structure of CumC.
CumD
In addition to the previously reported two complex
structures of CumD (acetate and isobutyrate), wecollected datasets of 6 complex structures (propionate, nbutyrate, n-valerate, isovalerate, (S)-2-methylbutyrate,and benzoate) at BL-6A and BL-18B [4].
Superimposition of the complex structures revealed that
benzoic acid was bound in a significantly different
direction compared with others, providing the structural
basis of the strict substrate specificity of CumD enzyme.
Moreover, the crystal structure of a mutant enzyme
showing high activity was determined using thediffraction data collected at BL-6A [5].

References[1] H. Aoki et al., J. Ferment. Bioeng. 81, 187 (1996).
[2] S. Fushinobu et al., Protein. Sci. 11, 2184 (2002).
[3] X. Dong et al., J. Bacteriol. 187, 2483 (2005).
[4] S. Fushinobu et al., Biosci. Biotechnol. Biochem. 69,491 (2005)[5] S.-Y. Jun et al., manuscript in preparation.
* asfushi@mail.ecc.u-tokyo.ac.jp