Life science
Medical & healthcare, Drug development

Structures and reaction mechanisms of posttranslational modification
enzymes targeting biosynthesis of novel functional peptides
Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research (SANKEN)
Associate Professor Toshihide Okajima
Researchmap https://researchmap.jp/read0068652?lang=en

Life science

Abstract
Specific chemical modification of proteins and peptides newlytranslated from genes, posttranslational modification, is essentialfor their functions and regulation. We focus the posttranslational
modifications of enzyme proteins including generation of intermolecular crosslinks and quinone cofactor. Our aim is to apply thesemodification enzymes for constructing a new platform for drug development and novel functional peptides.

▍ Background & Results
Quinohemoprotein amine dehydrogenase, derived from many
Gram-negative bacteria, catalyzes oxidative deamination of various aliphatic primary amines for assimilation. The smallest γ-subunit (QhpC) in three subunits αβγ of QHNDH has two uniqueposttranslational modifications: three thioether crosslinks and acovalently bound quinone cofactor, cysteine tryptophylquinone(CTQ). The complex modification suggests the multi-step mechanism involved in several modification enzymes. Based on ourprevious studies, QhpC is translated as a precursor form including a 28-residue leader peptide at N terminal and forms a ternarycomplex with QhpD and QhpG. A radical S-adenosyl-L-methionine(SAM) enzyme QhpD forms three thioether crosslinks in QhpC andsequentially FAD-dependent oxygenase QhpG di-hydroxylates aCTQ precursor, Trp residue of QhpC, in the complex. Followed withremoval of the leader peptide with a serine proteinase QhpE, themodified QhpC is transported to periplasm where CTQ is finallygenerated. As for QhpG, our group recently determined its X-ray
crystal structure and clarified details of the reaction mechanism
and the interaction with the substrate QhpC. QhpG is found be an
atypical single-component oxygenase, catalyzing dihydroxylationof an unmodified Trp residue in the protein substrate. We are able
to reconstitute these multi-step modification reactions without thelast step in vitro. QhpD reaction can generate a multi-loop or single
cyclic peptide, and QhpG is applicable to generate a reactive sitesuch as dihydroxyl Trp and a tryptophyl quinone. It is expected thatthese enzymes are applicable as tools to construct novel functional
peptides.

▍ Significance of the research and Future perspective
Recently, middle-size biomolecules including macro cyclic peptides with various functions are paid to attention as a new drug
framework because they have high affinity toward a target molecule and membrane permeability such as low molecular weightcompounds. Peptide modification enzymes shown in the present
work can be used for tools to develop new drugs or functional molecules on the cyclic peptides. Enzyme-based synthesis is possible
under ambient temperature and pressure, and attains low environmental loading, agreeing with a concept of green chemistry.

X-ray crystal structure of quinohemoprotein amine dehydrogenase and multiple
posttranslational modifications of QhpC.

Presumed model for ternary complex formed among radical SAM enzyme QhpD,FAD-dependent oxygenase QhpG, and substrate peptide QhpC.

P a t e n t
Oozeki, Toshinori; Nakai, Tadashi; Okajima, Toshihide et al. Functional and structural characterization of a flavoprotein monooxygenase essential forbiogenesis of tryptophylquinone cofactor. Nature Communications. 2021; 12(1): 933. doi: 10.1038/s41467-021-21200-9
Nakai, Tadashi; Tanizawa, Katsuyuki; Okajima, Toshihide et al. The radical S -adenosyl- L-methionine enzyme QhpD catalyzes sequential formation of
intra-protein sulfur-to-methylene carbon thioether bonds. J. Biol. Chem. 2015; 290(17): 11144-11166. doi: 10.1074/jbc.M115.638320
URL https://resou.osaka-u.ac.jp/ja/research/2021/20210210_2
K e y w o r d posttranslational modification, cyclic peptides, cofactor, thioether bondTr e a t i s e