微生物に見出した新規で多様なアミド結合形成酵素の網羅的解析と利用
大利 徹 （北大院工）Abstract
We identified a novel peptide ligase (PGM1) involved in pheganomycins biosynthesis. The enzyme activates a carboxyl group of an
N-amidino-phenylglycine derivative by phosphorylation in the presence of ATP and catalyzes the amide bond formation between the Namidino-phenylglycine derivative and ribosomally synthesized peptides. This is the first example of an ATP-grasp enzyme catalyzing
the amide bond formation with peptides as nucleophiles. We identified gene clusters containing a peptide ligase ortholog inactinobacteria by BLAST analysis. These gene clusters were heterologously expressed in Streptomyces lividans and Streptomyces
albus to probe the function of the orthologs. We detected a specific metabolite in culture broth of the transformant. We also identified anovel ATP-grasp enzyme, which is involved in peptidoglycan biosynthesis and catalyzed ligation between L-Glu and UDP-MurNAc-LAla. Besides, a novel epimerase catalyzing the epimerization of the terminal L-Glu of the product was also identified.

Background
Reaction mechanism of PGM1

Structure of pheganomycins (PGMs)‐ Produced by
Streptomyces cirratus‐ Exhibit anti‐
Mycobacterium activity

H
N‐NVKDR

t‐Leu(Val)

M. Noike et al., Nat Chem Biol 11, 71, (2015).

Biosynthetic gene cluster of PGMs

Lyspeptidase

MEREIVWTEIEESDLAAVVSASNVKDGPTVSSSNVKDR (38 amino acids)
DpgA DpgB

hyp

DpgC

amino‐trans. P450
ABCtransportertransporter

amidino‐trans.

ATP‐grasp ligase
Nocardiopsis baichengensis
Trphalogenase

P450

5 kbp DNA fragments

amidino‐trans.

hyphypmV600
Plasmid library
construction

acetyl‐trans.
flavoprotain

Transformation ofD‐Glu‐auxotrophic
E. coli mutant(WM335)

100control
2224

Retention time

26min

MS spectrum of peak 1[M+H]+214.35
intensity

Cultivation

Cl‐containingcompound

High yield

UV 340 nm

FDAA↓[M+H]+

1 mM

ATP

10 mM

β‐Mercaptoethanol

2.5 mM

MgCl220 mM(NH4)2SO425 mM

Tris‐HCl (pH 8.0)

100 mM

L‐Ala std.
D‐Glu std.
L‐Glu std.

5 µg/ml

Product

ESI negative

UV 340 nm FDAA

D‐Ala std.

Boiled XOO_1319

200300m/z

500400600Hydrolysis
XOO_1319

OH

XOO_1319 &1320

L‐Ala std.

Derivatization

D‐Glu std.

Cl
XOO_1320

L‐Glu std.

NMR analysis

ProductUDP‐MurNAc‐
L‐Ala‐L‐Glu

In vitro analysis

Chiral analysis
D‐Ala std.

UDP‐MurNAc‐L‐Ala‐L‐Glu

XOO_1319 or XOO_1320

216.430

UV 260 nm

UDP‐MurNAc‐L‐Ala

Retention time [min]Plasmid extraction& sequencing

HPLC/LC‐MS Analysis

Retention time [min]With L‐Glu
1

UDP‐MurNAc‐L‐Ala‐L‐Glu

Assay mixture

20

UDP‐MurNAc‐L‐Ala‐D‐Glu
MW = 879.1

Low yield

XOO_1319 epimerase
assaytransformant

2000

With D‐Glu

1

UDP‐MurNAc‐L‐Ala1 mM
ATP
10 mM
β‐Mercaptoethanol
2.5 mM
20 mM
MgCl225 mM(NH4)2SO4
Tris‐HCl (pH 8.0)
100 mM
D‐Glu or L‐Glu (control)
XOO_1320
5 µg/ml300

tsr

Streptomyces lividans
orStreptomyces albus

XOO_1320 Glu ligase assay
Assay mixture

1

500
400

pWHM3

regulator

Peptideligase

HPLC analysis

tsr

DpgC

49%
MFS
transporter identity CH3

hyp

pUC19

pWHM3

Transformation

DpgA

Peptidoglycan

enoyl‐CoA
hydratase

DpgB

CH3

Digested by Sau3AI

Cloning

peptide ligase (PGM1)

AU

Peptidoglycan
X. oryzae genomic DNA

PCR

radical SAM(CH3)Results

MethodATP‐graspligase

CH3AU

Asn

H2N‐NVKDR

peptide ligase(PGM1)R= OH
Asp
Asp‐Arg
Asp‐Gly‐Pro‐Thr
N‐amidino‐phenylglycine

Ribosomallyformed

HN

CH3O

Retention time [min]R. Feng, et. al. J. Am. Chem. Soc., 139, 4243 (2017)