Photon Factory Activity Report 2017 #35 (2018)

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

Crystal structures of the enzymes involved in novel lysine biosynthetic pathway usingamino-group carrier protein
Takeo TOMITA1, Ayako YOSHIDA1, Masanobu ARITA1, 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 ofevolutionary origins of the amino acid biosynthetic
pathways. Thus, to reveal the structural basis of thislysine biosynthetic pathway is important for the further
understanding of LysW-mediated amino acid biosyntheticmachinery.
We have determined the crystal structures of thebiosynthetic enzymes (LysX, LysZ, and LysY) incomplex with LysW or its derivatives, whereas the crystal
structures of LysJ and LysK, which catalyze the last twosteps, in complex with LysW have not been determined.
For LysK, crystal structure in complex with lysine wasdetermined and the mutational studies proposed therecognition mechanism of LysW by LysK. LysJ functions
as aminotransferase to produce LysW-γ-Lys from LysW-γAAA semialdehyde in PLP-dependent manner. To
understand the recognition mechanism of LysJ for LysW
derivatives, we performed the crystallization screening for
LysJ·LysW derivatives complex and the mutational
studies for LysJ.
2 Materials and Methods
Purification of recombinant proteins
LysJ and its mutants was overexpressed in E. coli
BL21-Codon-Plus (DE3)-RIL as a host. From cell lysate
prepared by sonication, LysJ was puridied through heat2+treatment, Ni -NTA column, and gel filtration
chromatography (Superdex 200) for crystallization.
LysW-γ-Lys, which can be a substrate of LysJ, wasproduced using E. coli BL21 (DE3) harboring theexpression vectors for LysW, LysX, LysZ, LysY, and LysJ
with 5 mM AAA supplemented in the culture broth. Fromcell lysate prepared by sonication, LysW-γ-Lys was
purified through heat treatment, anion exchange
chromatography (DE52, monoQ), and gel filtration
chromatography (Superdex 30). LysW was also preparedby the same procedure. LysW-γ-AAA was produced by
the enzyme reaction by LysX using LysW and AAA assubstrates, and purified in the same way as LysW.
Activity assay for LysJ and its mutants
To reveal the recognition mechanism for LysW-γ-Lys inLysJ, we introduced mutations for positively-charged
residues around the active site, especially residues
conserved in LysJ but not in ArgD, which is a paralogous
protein in the general (not LysW-mediated) arginine
biosynthesis. Activity of these mutants were measured
using LysW-γ-Lys and Nα-acetyllysine.
Co-crystalization of LysJ·LysW derivatives
The condition for co-crystallization of LysJ·LysW-γLys or LysW derivatives was screened with Crystal screen
I and II, Wizard classic I, II, and III, and PEG/ION by
hanging drop vapor diffusion method at 20 ˚C.
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 or LysW. The optimization of thecrystallization condition allowed the diffraction at 2.5-3 Å,
however, the diffraction quality was not good enough toobtain the data set. Further optimization of thecrystallization or cryo-protectant condition is necessary.
Recognition mechanism of LysJ for LysW-γ-Lys
Some of the mutations around the active site reduced
the LysJ activity against LysW-γ-Lys but not against Nαacetyllysine. This indicated that these residues are notimportant for the catalysis but are involved in therecognition of LysW moiety of LysW-γ-Lys. Theseresidues are located around the positively-charged surfaceof LysJ, suggesting that these residues might beresponsible for the recognition of the globular domain ofLysW.
* umanis@mail.ecc.u-tokyo.ac.jp