Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

5A,6A,NW12/2003G134

Crystallographic analysis of LysN, α-aminoadipateaminotransferase, from T.
thermophilus HB27
1
Takeo Tomita1, Takashi Miyazaki1, Tomoharu Miyagawa, Tomohisa Kuzuyama1, Makoto
1
Nishiyama*
1
Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Introduction
We previously cloned lysN gene from Thermus
thermophilus HB27 as a homolog of mammalian
Kynurenine aminotransferase II (KatII) and characterized
as a gene playing a major role in the fourth reaction(transamination reaction using 2-oxoadipate as an amino
acceptor to yield a-aminoadipate) of AAA pathway forlysine biosynthesis [1]. LysN recognizes not only 2oxoadipate, an intermediate of lysine biosynthesis but
also 2-oxoisocaproate, 2-oxovalerate and 2-oxomethylvalerate, intermediates of leucine, valine andisoleucine biosynthesis, respectively, along withoxaloacetate, a compound in the TCA cycle, as anaminoacceptor. Althouth these results suggests multiple
roles in LysN in several cellular metabolic pathways
including lysine and branched-chain amino acidbiosynthesis, structural basis for the broad range substratespecificity of LysN remained to be elucidated. Therefore,
we conducted crystallographic analysis of LysN.
Materials and Methods
E.coli BL21-Codon-Plus (DE3)-RIL cells harbouring
pETLysN7 were cultured in 800 ml of 2x YT medium
containing 50 µg/ml kanamycin and 30 µg/ml
chloramphenicol. When the culture had reached an
OD600 of 0.5, IPTG (final concentration, 0.1 mM) wasadded. The culture was continued at 25°C for an
additional 12 h after the induction. E. coli (pETLysN7)
cells (wet weight, 4.5 g) collected from the 800 ml culture
were suspended in 27 ml buffer I (20 mM potassium
phosphate buffer, pH6.5, 0.5 mM EDTA ) and disrupted
by sonication. The supernatant prepared by centrifugation
at 40 000 g for 20 min was heated at 80°C for 20 min, anddenatured proteins from E.coli cells were removed bycentrifugation at 40 000 g for 20 min. Supernatantfractions were applied to an anion-exchange column (DE52; Whatman), pre-equilibrated with buffer I and eluted
with buffer I containing 0.1 M NaCl. After the addition ofammonium sulfate to a final concentration of 65%
saturation to active fractions, the resultant precipitate wascollected by centrifugation at 40 000 g for 30 min. The
precipitated proteins were solubilized with buffer I andapplied onto a Hi-load 26/60 Superdex 200 prep-grade
column (Amersham-Pharmacia Biotech) equilibrated withbuffer I containing 0.1 M NaCl to yield the purified
preparation. Protein concentration was determined by themethod of Bradford using a protein assay kit (NipponBio-Rad).

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Results and Discussion
Crystallization
Following the purification, LysN was concentrated upto
5 mg/ml. Crystals of LysN were obtained by the vapor
diffusion method with a reservoir solution containing 813% (w/v) polyethylene glycol (PEG) 6000 and 100 mM
MES-NaOH (pH6.0-6.5) within 1-2 day at 20°C.
Data collection and processing
Before data collection, the crystals were transferred toa reservoir solution finally supplemented with acryoprotectant, 25% PEG 400 (w/v) by increasing theconcentration of cryoprotectant to 25% by 5% in eachstep, with equilibration for 1 minute between the steps.
Crystals were flash-frozed in a cold N2 stream from aliquid-nitrogen cryostat (Rigaku). All the data collection
were carried out at 100 K. Diffraction dataset wascollected with an ADSC Quantum CCD detector on theBL5A station at Photon Factory in High Energy
Accelerator Research Organization (KEK). Data
processing and scaling were performed using HKL2000.
Statistics of data collection was summerized inTable1.Crystal structure determination is now underway.
Table1: Statistics of data collection
CrystalA

Space group

P21

Cell dimensions (Å)

a=55.95,b=167.64,
c=97.87

Resolutionshell)(Å)(outer2.26 (2.26-2.34)

Rmerge (%)

12.2

I/σ (I)

19.4 (4.9)

Completeness (%)

99.3 (100.0)

References[1] T. Miyazaki et al., Microbiology, 150, 2327 (2004).
*umanis@mail.ecc.u-tokyo.ac.jp