Photon Factory Activity Report 2019 #37 (2020)
BL-5A, BL-17A, AR-NW12A, AR-NE3A/2019G548

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

Takeo TOMITA1,2, Ayako YOSHIDA1, Sumire KUROSAWA1, and Makoto NISHIYAMA1,2,*Biotechnology Research Center, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657,
Japan
2
CRIIM, 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, bynonribosomal 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 thefurther understanding of AmCP-mediated amino acidbiosynthetic machinery. Here, we describe the trial todetermine the crystal structures of enzymes involved inthese pathways to reveal the structural basis of thisunprecedented biosynthetic pathway including AmCP.
2 Experiment
Purification of recombinant proteins
Complex of OrfX and OrfY which are enzymes involved
in biosynthesis of vazabitide A, and OrfZ which is an
enzyme involved in biosynthesis of s56-p1 were
overexpressed in E. coli Rosetta2 (DE3) and E. coli BL21Codon-Plus (DE3)-RIL as the host, respectively. From celllysates prepared by sonication, Complex of OrfX and OrfYwere purified through Ni2+-NTA column and OrfZ waspurified through strep-tactin column, following by gel
filtration
chromatography(Superdex200)
forcrystallization.
Crystallization of complex of OrfX and OrfY, and OrfZ
The condition for crystallization of Complex of OrfX
and OrfY, and OrfZ 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 the crystallization condition was also
conducted by modifying the concentration and pH.
3 Results and Discussion
X-ray diffraction analyses of complex of OrfX and OrfY
We found the crystallization condition of complex ofOrfX and OrfY. We carried out X-ray diffraction analyses,and obtained data set at 1.9 Å resolution. Determination ofthe structure of them is now in progress by means of singlewavelength anomalous dispersion (SAD) method.
X-ray diffraction analysis of OrfZ
We have not found the crystallization condition of OrfZ.
Crystallization of OrfZ with different construction is nowin 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