Photon Factory Activity Report 2022 #40 (2023)

BL-5A, BL-17A, AR-NE3A/2021G672

Crystal structures of enzymes involved in the biosynthetic pathways using aminogroup carrier protein, AmCP
Sumire KUROSAWA1, Ayako YOSHIDA1,2, Takeo TOMITA1,2 and Makoto NISHIYAMA1.2*
1
AgTECH, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2CRIIM, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
1 Introduction
The lysine biosynthetic pathway is classified into twotypes; the diaminopimelate (DAP) pathway in bacteria andplants, and the α-aminoadipate (AAA) pathway in fungi
and yeast. We found that a bacterium, Thermus
thermophilus synthesizes lysine via a novel AAA pathway
using a small acidic protein called LysW. T. thermophilus
uses LysW protein to protect α-amino group of AAA to
avoid self-cyclization of the intermediate. Our previous
studies including crystallographic analyses revealed that
LysW functions not only as an amino-group protecting
group but also as a carrier protein interacting with eachenzyme electrostatically. Therefore, we hereafter call
LysW as amino-group carrier protein (AmCP). Since our
discovery of AmCP-mediated lysine biosynthesis throughAAA in T. thermophilus, many microorganisms including
thermophilic bacteria and archaea synthesize lysine andarginine by a similar pathway, indicating that this pathway
is one of evolutionary origins of the amino acidbiosynthetic pathways.
Furthermore, we have found that Streptomyces uses
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 subsequent
modification enzymes. The biosynthetic gene clusters ofazinomycin B and ficellomycin, which also possess
azabicyclo-ring moiety, contain DADH biosynthetic genes
including AmCP gene, suggesting that these compounds
are also biosynthesized via AmCP. Since an aziridine ringin this azabicyclo-ring is the key moiety for DNA
alkylation, we are interested in the mechanism of this ringformation.
Taken together, the elucidation of the structural bases ofthis unprecedented biosynthetic pathway is important for
the further understanding of AmCP-mediated amino acidbiosynthetic machinery. Here we present the structural
analysis of the enzymes involved in the DADH
biosynthesis and aziridine-ring formation.
2 Experiment
Purification of recombinant proteinsVzb10/11 and AziU3/U2 complex, which are enzymes
involved in aziridine ring formation in biosynthesis ofvazabitide A and azinomycin B, respectively, and Fic25

involved in the DADH biosynthesis in ficellomycin
biosynthesis, were overexpressed in E. coli Rosetta2(DE3)and BL21-Codon-Plus (DE3)-RIL as hosts. From celllysates prepared by sonication, these proteins were purified
using Ni2+-NTA column, followed by gel filtration
chromatography (Superdex 200) for crystallization.
Crystallization
The crystallization conditions for each protein (proteincomplex) were screened with Crystal screen I and II,
Wizard classic I, II, and III, and PEG/ION screen byhanging drop vapor diffusion method at 20ºC.
Optimization of the crystallization conditions were also
performed by changing the concentration of precipitants
and pH.
3 Results and Discussion
Structure of AziU2/U3 complex
We successfully determined the structure of AziU2/U3complex at 1.75 Å resolution by SAD method. The
complex forms hetero-tetramer with a novel fold. We also
determined the structure of AziU2/U3 in complex with its
substrate at 1.8 Å resolution. The structural informationand the activity assay using variants suggested the catalytic
mechanism for unprecedented aziridine-ring formation [2].
Structure of Fic25
We crystallized Fic25, which is an aminotransferaseinvolved in the production of DADH in the ficellomycin
biosynthesis, and successfully determined the structure in
apo, holo-forms and reaction intermediate-bound form at
1.80, 1.93, and 1.82 Å resolution, respectively. DADHs
biosynthesized in ficellomycin and vazabitide A havedifferent stereochemistry. The structure with PLP-substrate
adduct and the activity assay using variants of Fic25 andVzb9, which is a homologous protein in vazabitide A
biosynthesis, revealed the mechanism to synthesize
stereoisomers of DADH [3].
References[1] Hasebe, F. et al., Nat. Chem. Biol. 12, 967 (2016).
[2] Kurosawa, S., J. Am. Chem. Soc. 144, 16164 (2022).
[3] Kurosawa, S., ACS Chem. Biol. 18, 385 (2023).
* umanis@g.ecc.u-tokyo.ac.jp