Photon Factory Activity Report 2016 #34 (2017)

BL-5A, BL-17A, AR-NW12A, AR-NE3A /2015G552

Crystal structures of the enzymes involved in novel lysine biosynthetic pathway
using amino-group carrier protein
Takeo TOMITA1, Ayako YOSHIDA1, Satomi Fujita1, and Makoto NISHIYAMA*1
1
The Univ. of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
1 Introduction
Lysine biosynthetic pathway is classified into twotypes; diaminopimelate (DAP) pathway in bacteria andplants, and α-aminoadipate (AAA) pathway in fungi andyeast. We previously found that a bacterium, Thermus
thermophilus synthesizes lysine via a novel type of AAA
pathway using an amino-group carrier protein called
LysW. 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
proteinandourpreviousstudies
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 throughthe electrostatic interactions. Since our discovery of
LysW-mediated lysine biosynthesis through AAA in T.
thermophilus, evidences have mounted that many
microorganisms including thermophilic bacteria andarchaea synthesize lysine and also arginine by a similar
pathway, indicating that this pathway is one of origins of
the amino acid biosynthetic pathways. Thus, to reveal thestructural basis of this lysine biosynthetic pathway isimportant for the further understanding of LysW-mediatedamino acid biosynthetic machinery.
We have determined the crystal structures of thebiosynthetic enzymes (LysX, LysZ, and LysY) incomplex with LysW or its derivatives, whereas the crystalstructures of LysJ and LysK, which catalyze the last twosteps, in complex with LysW have not been determined.
LysJ functions as aminotransferase to produce LysW-γLys, and LysK is a metalloprotease hydrolyzing LysW-γLys to release lysine. Previously, we determined thecrystal structure of LysK·lysine complex. To understandthe recognition mechanism of these enzymes for LysW
derivatives, we performed the crystallization screening
for both enzymes and the mutational studies for LysK.
2 Materials and Methods
Purification of recombinant proteins
LysJ and LysK and its mutants were overexpressed inE. coli BL21-Codon-Plus (DE3)-RIL as the hosts. From
cell lysate prepared by sonication, LysJ was puridied
2+through heat treatment, Ni -NTA column, and gel
filtration chromatography.
LysK
waspurified
through heat treatment, Strep-tactin column, andgel filtration chromatography.

LysW-γ-Lys was produced using E. coli BL21 (DE3)
harboring the expression vectors for LysW, LysX, LysZ,
LysY, and LysJ using 0.1 mM IPTG for induction
supplemented with 5 mM AAA. From cell lysate preparedby sonication, LysW-γ-Lys was purified through heat
treatment, anion exchange chromatographies, and gel
filtration chromatography.
Activity assay for LysK mutants
To reveal the recognition mechanism for LysW in LysK,
we introduced mutations for positively-charged residues
around the active site and the residues consisting the cleft
toward the active site. Activity of these mutants weremeasured using LysW-γ-Lys and CT10-γ-Lys, which isthe synthetic oligopeptide containing the C-terminal 10
residues of LysW fused with lysine.
Co-crystalization of LysJ/LysW-γ-Lys
The condition for LysJ/LysW-γ-Lys co-crystalization
was screened with Crystal screen I and II, Wizard classic
I, II, and III, and PEG/ION by hanging drop vapor
diffusion method.
3 Results and Discussion
Crystals of LysJ/LysW-γ-Lys
We found the crystallization condition of LysJ in which
PEG3350 was used as a precipitant in the presence ofLysW-γ-Lys. We analyzed the X-ray diffraction data, but
it only diffracted at 5-6 Å resolution. Further optimizationof the crystallization condition is necessary to obtain thedata set.
Recognition mechanism of LysK for LysW-γ-Lys
The mutations at the cleft for the active site reduced theLysK activity, indicating that these residues wellrecognize the C-terminal extension domain and fusedlysine. However, among the five positively-chargedresidues, only two mutants reduced their activity. Using
CT10-γ-Lys as a substrate, the activity of these mutants
was less decreased than the activity toward LysW-γ-Lys,suggesting that these residues are involved in therecognition of the globular domain of LysW. The docking
model of LysK bound with LysW-γ-Lys suggested that afew specific interactions were observed between LysKand the globular domain of LysW. We assumed that thepositively-charged area of LysK functions to attract
LysW-γ-Lys near the active site, but weakly interact withthe LysW derivative, as reported previously for otherbiosynthetic enzymes complexes with LysW.
* umanis@mail.ecc.u-tokyo.ac.jp