Photon Factory Activity Report 2018 #36 (2019)

BL-5A, BL-17A, AR-NW12A, AR-NE3A/2017G574

Crystal structures of enzymes involved in novel lysine biosynthetic pathway usingamino-group carrier protein
1

Takeo TOMITA1, Ayako YOSHIDA1, Sumire KUROSAWA1, and Makoto NISHIYAMA1,*
Biotechnology Research Center, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657,
Japan

1 Introduction
Lysine biosynthetic pathway is classified into two types;
diaminopimelate (DAP) pathway in bacteria and plants,and α-aminoadipate (AAA) pathway in fungi and yeast.
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 of thepathway in T. thermophilus are similar to those of fungi,
the latter part is composed of the enzymes similar to thosein arginine biosynthesis. Unlike in the arginine
biosynthesis, T. thermophilus uses LysW protein to protect
α-amino group of AAA. LysW is highly acidic protein andour previous studies including crystallographic analyses
revealed that LysW functions not only as an amino-groupprotecting group but also as a carrier protein by interactingwith each enzyme through the electrostatic interactions.
Therefore, we hereafter call LysW as amino-group carrier
protein (AmCP). Since our discovery of AmCP-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 evolutionary origins of theamino acid biosynthetic pathways.
Recently, we found that a kind of Streptomyces used
AmCP in the biosynthesis of secondary metabolites whichcontaining non-proteinogenic amino acid (2S, 6R)diamino-(5R, 7)-dehydroxy-heptanoic acid, DADH [1].
DADH is incorporated into a novel peptide metabolite,
vazabitide A, featuring an azabicyclo-ring structure, by
nonribosomal peptide synthetases and successive
modification enzymes.
We also found that AmCP was also used for thebiosynthesis of a compound, s56-p1, produced by anotherstrain of Streptomyces [2]. s56-p1 contains a uniquenitrogen-nitrogen bond (hydrazine moiety) fused with theDADH-derivative moiety. We identified the unprecedented
machinery for hydrazine moiety in the strain and also
found this machinery is widespread across several bacterial
phyla, highlighting the overlooked potential of bacteria tosynthesize hydrazine.
Thus, to reveal the structural bases of these unprecedented
biosynthetic pathway is important for the further
understanding of AmCP-mediated amino acid biosyntheticmachinery. Here, we describe the trial to determine thecrystal structures of enzymes involved in these pathways toreveal the structural basis of this unprecedented
biosynthetic pathway including AmCP.
2 Experiment
Purification of recombinant proteins
OrfX which is an enzyme involved in biosynthesis ofvazabitide A, and OrfY which is an enzyme involved inbiosynthesis of s56-p1, and the truncated type of OrfY,OrfYtr, were overexpressed in E. coli BL21-Codon-Plus(DE3)-RIL as the host. From cell lysates prepared bysonication, OrfX, OrfY, and OrfYtr were purified through2+Ni -NTA column, and gel filtration chromatography(Superdex 200) for crystallization.
Crystallization of OrfX, OrfY, and OrfYtr
The condition for crystallization of OrfX, OrfY, andOrfYtr were screened with Crystal screen I and II, Wizard
classic I, II, and III, and PEG/ION by hanging drop vapor
diffusion method at 20 C̊. Optimization of thecrystallization condition was also conducted by modifying
the concentration and pH.
3 Results and Discussion
X-ray diffraction analyses of OrfX and OrfY
We found the crystallization condition of OrfX and OrfY.
We carried out X-ray diffraction analyses, however, thediffraction qualities were not good enough to obtain thedata set. Further optimization of the crystallization or cryoprotectant condition is necessary.
X-ray diffraction analysis of OrfYtr
We found the crystallization condition of OrfYtr. We
carried out X-ray diffraction analysis, and obtained data set
at 1.8 Å resolution. By means of single-wavelength
anomalous dispersion (SAD) method using anomalous
signal of metal ion bound to OrfYtr, we determined initialstructure of OrfYtr. Refinement of the structure of OrfYtr
is now in progress.
References[1] Hasebe, F. et al., Nat. Chem. Biol. 12, 967 (2016).
[2] Matsuda, K., J. Am. Chem. Soc. 140, 9083 (2018).
* umanis@mail.ecc.u-tokyo.ac.jp