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

6A, 18B/2004G152

Crystal structure determination of DtsR1, a carboxyltransferase subunit ofacetyl-CoA carboxylase in Corynebadterium glutamicum
Ryo Natsume1, Minoru Yamada1,2, Miki Senda1, Toshiya Senda*3
1
JBIRC, JBIC, Aomi 2-42, Koto-ku, Tokyo 135-0064, Japan
2
Tokyo Denki Univ., 2-2 Kanda-Nishiki-cho, Chiyoda-ku, Tokyo 101-8457, Japan,
3BIRC, AIST, Aomi 2-42, Koto-ku, Tokyo 135-0064, Japan
Introduction
Acetyl-CoA carboxylases (ACCs) catalyze the firstcommitted step of fatty acid biosynthesis. Although ACC
is an essential enzyme (complex) in every organism, thestructure-function relationship of ACC remains unclear.
ACC from Corynebacterium glutamicum is a multisubunit complex composed of AccBC and DtsR1. As thefirst step for elucidating the structure-function
relationship of ACC, we initiated the crystallographic
analysis of DtsR1. DtsR1 is the core catalytic β-subunitof ACC from C. glutamicum, which catalyzes thetranscarboxylation between biotin and acetyl-CoA.
DtsR1 was crystallized by the sitting-drop vapordiffusion method using PEG 6000 as a precipitant. The
approximate dimensions of the obtained crystals were30.07x0.07x0.03mm . Here we report the crystal structure
determination of DtsR1.
Results
Data collection and processing
All the data collections were carried out at 100K. The
crystals were soaked in a cryoprotectant solution for afew seconds and flash-frozen in a cold N2 stream from aliquid-nitrogen cryostat (Rigaku). Diffraction data werecollected using an ADSC Quantum CCD detector at NW12 of PF-AR.
Data processing and scaling were performed using
HKL2000 (Table 1). The crystal belongs to the spacegroup R32 and likely contains three subunits in theasymmetric unit.
Table 1: Statistics of data collection
IIIa = b = 204.25, a = b = 204.15,
Cell dimensions (Å)
c = 234.00
c = 233.72
Matthews
2.62
2.61
3 -1
coefficient (Å Da )Solvent content (%) 53.01
52.90
Resolution (Å)
48.8-3.15
50.04-2.63
a(outer shell)(3.26-3.15)(2.72-2.63)
bRmerge (%)
9.3 (50.1)
12.0 (87.7)
Completeness (%)
99.8 (99.8)
93.3 (58.0)
Multiplicity7.8 (7.7)
3.4 (1.8)
a: Statistics for the highest shell are given in parenthesesNb: Rmerge = ΣhΣi |I(h)i-I(h)| / Σh(N + I(h)i)
Crystal250

Users' Report

Crystal structure determination
Molecular replacement was performed with theprogram MOLREP in the CCP4 suite using a 12S
monomeroftranscarboxylase
derivedfrom
Propionibacterium shermanii (PDB code; 1ON3) [1] as asearch model, which shows 49% amino acid sequence
identity with DtsR1. The data from crystal I was used forthe calculation. Because there were no significant peaksin the cross-rotation function, translation functions werecalculated using each of the first 30 peaks of the rotation
function. We then found three subunits in the asymmetricunit without unfavorable molecular contacts in the crystal
packing and started model building with the threesubunits as the initial model. The crystal packing strongly
suggests that the DtsR1 is a ring-shaped homo hexamer
with 32-point group symmetry, as found in other relatedproteins. One subunit in the asymmetric unit forms ahexamer with its symmetry-related subunits. The othertwo subunits in the asymmetric unit, which make a noncrystallographic dimer, form another hexamer with theirsymmetry-related subunits.
Crystallographic refinement is in progress using thedata of crystal II at 2.7Å resolution.
References[1] P. R. Hall et al., EMBO J. 22, 2334 (2003).
* tsenda@jbirc.aist.go.jp