. 2018 May 17;8:7810. doi: 10.1038/s41598-018-26179-w

Table 2.

Analysis of limazepine biosynthetic gene cluster.

Protein	Homologous proteins (from PBDs and phenazines)					Phenazines	Identity/similarity to homologue with*	Function of limazepine biosynthetic protein
Limazepine	Tomaymycin	Anthramycin	Porothramycin	Sibiromycin	Tilivalline	Phenazines	Identity/similarity to homologue with*	Function of limazepine biosynthetic protein
outside biosynthetic gene cluster
ORF1	—	—	—	—	—	—	49/60	chromosome segregation protein Spo0J, highest homology to: WP_020636716.1* (primary metabolism)^C
PBD assembly
Lim1	TomA¹⁸*	ORF21	POR20	SibE¹⁷	NpsA, ThdA	—	50/62	NRPS (anthranilate precursor activation)^A
Lim2	TomB¹⁸*	ORF22	POR21	SibD	NpsB	—	50/61	NRPS (APD precursor activation and condensation)^A
biosynthesis of anthranilate moiety
Lim3	TomC*	—	—	—	AroX	PhzC	62/73	DAHP synthase^B
Lim4	—	—	—	—	DhbX*	—	50/59	DHHA oxidoreductase^B
Lim5	—	—	—	—	IcmX*	PhzD	53/65	isochorismatase (pyruvate removal from ADIC)^C
Lim6	TomD* TomP	—	—	—	AdsX	PhzE	56/65	ADIC synthase^C
Lim7	TomE*	—	—	—	—	—	61/71	oxidoreductase (hydroxylation)^B
Lim8	TomF*	—	—	—	—	—	83/90	monooxygenase (hydroxylation)^B
Lim9	TomG*	—	—	—	—	—	62/73	methyltransferase (O-methylation)^B
biosynthesis of APD
Lim10	TomH*	ORF12⁴⁸	POR13	SibV⁴⁸	—	—	65/73	L-DOPA-2,3-dioxygenase (oxidative cleavage of L-DOPA)^A
Lim11	TomI	ORF13⁴⁹	POR14	SibU*	—	—	49/62	tyrosine hydroxylase (synthesis of DOPA)^A
Lim12	TomJ*	ORF14	POR15	SibT	—	—	67/78	F-420 dependent reductase (double bond reduction)^D
Lim13	TomK*	ORF15	POR16	SibS	—	PhzF	47/56	Isomerase (double bond isomeration)^D
Lim14	TomL*	ORF6⁵⁰	POR11	SibY	—	—	66/76	ƴ- glutamyltransferase-like hydrolase (C-C bond cleavage)^A
resistance/regulation/unknown function
Lim15	TomM	ORF8*	POR3	SibF	UvrX	—	72/84	excinuclease ABC subunit A (resistance)^B
Lim16	—	—	—	—	—	—	96/98	LLM class flavin-dependent oxidoreductase, highest homology to: WP_051872335.1* (unknown function)^B
Lim17	—	—	—	—	—	—	61/76	MarR family DNA-binding transcriptional regulator, highest homology to: WP_086678485.1* (regulation)^B
Lim18	TomQ*	ORF24	POR5	—	—	—	67/79	amine oxidase (unknown function)^B
outside biosynthetic gene cluster
ORF2	—	—	—	—	—	—	70/79	cation:proton antiporter, highest homology to: WP_030262790.1* (primary metabolism)^B

^*Indicates the closest homologue of the respective Lim protein.

^AProposed based on elucidation of a homologue from PBD biosynthesis (reference included).

^BProposed based on sequence homology.

^CConfirmed in vitro in this study.

^DProposed based on indirect in vivo studies of lincomycin biosynthesis, which shares APD biosynthesis with PBDs¹⁴.