Photon Factory Activity Report 2014 #32 (2015) B
AR-NW12, AR-NE3, BL-5A /2013G619

Crystal structures of the enzymes involved in novel lysine biosynthetic pathway
using amino-group carrier protein
Takeo TOMITA1, Ayako YOSHIDA1, Tetsu SHIMIZU1, Satomi FUJITA1, Fumihito HASEBE1,and Makoto NISHIYAMA*1
1
The Univ. of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Introduction
Lysine biosynthetic pathway is classified into twogroups; diaminopimelate (DAP) pathway in bacteria andplants, and α-aminoadipate (AAA) pathway in fungi andyeast. We previously found that a bacterium, Thermus
thermophilus synthesize lysine via a novel type of AAA
pathway using an amino-group carrier protein calledLysW. Although the enzymes involved in the first half ofthe pathway in T. thermophilus are similar to those offungi, the latter part is composed of the enzymes similar
to those in arginine biosynthesis. Unlike in the arginine
biosynthesis, T. thermophilus uses LysW protein toprotect α-amino group of AAA. LysW is highly acidic
proteinand
ourpreviousstudies
including
crystallographic analyses revealed that LysW functions
not only as an amino-group protecting group but also as acarrier protein by interacting with each enzyme
electrostatically. Since our discovery of LysW-mediatedlysine biosynthesis through AAA in T. thermophilus,
evidences have mounted that many microorganisms
including thermophilic bacteria and archaea synthesize
lysine and also arginine by a similar pathway, indicatingthat this pathway is one of origins of the amino acidbiosynthetic pathways. Thus, to reveal the structural basisof this novel lysine biosynthetic pathway is important for
the further understanding of LysW-mediated amino acidbiosynthetic machinery.
To date, we have clarified the electrostatic interaction
between LysW and several enzymes involved in the AAApathway. Here we report the structural analysis about
LysK, which catalyzes the final step of this pathway.
LysK is a member of M20 metallopeptidase family, whichcatalyzes the release of lysine from LysW--Lys, which
produced by enzyme reactions from AAA, in the presenceof metal ions. Although some structures of M20 family
peptidases are determined, most of those structures aredetermined in the absence of substrates. Therefore,
structural determinants of substrate specificity of M20
family peptidases are not known due to the lack ofstructural information. As seen in other biosynthetic
enzymes in AAA pathway, the active site of LysK was
also predicted to be surrounded by positively-charged
residues to recognize the negatively-charged globular
domain of LysW. However, the experimental verificationby the crystal structure has not yet been performed until
now. To clarify the substrate recognition manner and theinteraction mechanism between LysK and LysW--Lys,

we performed crystallographic analysis of LysK andLysW complex from T. thermophilus HB27.
Materials and Methods
Purification of recombinant proteins
LysK was overexpressed in Escherichia coli BL21Codon-Plus (DE3)-RIL as a host using 0.1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) for induction. Fromcell lysate prepared by sonication, LysK was purified
throughheat
treatment,
Strep-Tactin
affinity
chromatography, and Superdex 200 gel filtration
chromatography.
LysW was overexpressed in E. coli BL21-Codon-Plus(DE3)-RIL as a host using 0.1 mM IPTG for induction.
Cells were harvested and sonicated to prepare the lysate.
LysW was purified through heat treatment, DE52 anion
exchange chromatography, and Superdex 30 gel filtration
chromatography.
Crystallization of LysK/LysW complex
Crystallization condition for
LysK/LysW cocrystallization was screened in the presence of lysine
using Crystal screen I and II, and Wizard classic I, II, andIII by hanging drop vapor diffusion method.
Results and Discussion
Through heat treatment and successive column
chromatographies, we could purify LysK and LysW.
We found the crystallization condition in whichammonium sulfate was used as a precipitant in thepresence of LysW and lysine. We collected the diffraction
data at 2.4 Å resolusion and determined crystal structureof LysK/lysine complex. Although we added LysW andlysine to the mixture, only lysine was bound to LysK.
Although LysK did not bind metal ions since thecrystallization condition did not contain any metals, thestructure revealed that LysK has metal-binding site
composed of same residues as other members of M20
metallopeptidase family. The structure also revealed
lysine-binding manner of LysK and suggested structural
determinants of substrate specificity of M20 family
enzymes. The surface region surrounding the active siteof LysK was occupied by positively charged residues,suggesting that LysK recognizes globular domain ofLysW through electrostatic interaction. We are now trying
to crystallize LysK/LysW--Lys complex to exemplify theinteraction manner between two proteins.
* umanis@mail.ecc.u-tokyo.ac.jp