Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803.
Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. Protox is encoded by the hemY gene in eukaryotes and by the hemG gene in many γ-proteobacteria, including Escherichia coli. It has been suggested that other bacteria possess a yet unidentified type of Protox. To identify a unique bacterial gene encoding Protox, we first introduced the Arabidopsis hemY gene into the genome of the cyanobacterium, Synechocystis sp. PCC6803. We subsequently mutagenized the cells by transposon tagging and screened the tagged lines for mutants that were sensitive to acifluorfen, which is a specific inhibitor of the hemY-type Protox. Several cell lines containing the tagged slr1790 locus exhibited acifluorfen sensitivity. The slr1790 gene encodes a putative membrane-spanning protein that is distantly related to the M subunit of NADH dehydrogenase complex I. We attempted to disrupt this gene in the wild-type background of Synechocystis, but we were only able to obtain heteroplasmic disruptants. These cells accumulated a substantial amount of protoporphyrin IX, suggesting that the slr1790 gene is essential for growth and Protox activity of cells. We found that most cyanobacteria and many other bacteria possess slr1790 homologs. We overexpressed an slr1790 homolog of Rhodobacter sphaeroides in Escherichia coli and found that this recombinant protein possesses Protox activity in vitro. These results collectively demonstrate that slr1790 encodes a unique Protox enzyme and we propose naming the slr1790 gene "hemJ."
Kato
Kazushige
K
Odawara Research Center, Nippon Soda Co., Ltd., Odawara 250-0280, Japan. k.katou@nippon-soda.co.jp
Tanaka
Ryouichi
R
Sano
Shinsuke
S
Tanaka
Ayumi
A
Hosaka
Hideo
H
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018390:Gene Targeting; D005798:Genes, Bacterial; D017343:Genes, Plant; D016254:Mutagenesis, Insertional; D009579:Nitrobenzoates / Q000494:pharmacology; D010802:Phylogeny; D050768:Protoporphyrinogen Oxidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D009579:Nitrobenzoates; D011994:Recombinant Proteins; D050768:Protoporphyrinogen Oxidase; C018425:acifluorfen
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8895-900
9238074
J Bacteriol. 1992 Jun;174(12):3953-63
1317844
Annu Rev Plant Biol. 2007;58:321-46
17227226
Science. 2000 Sep 8;289(5485):1724-30
10976061
Photosynth Res. 2001;70(1):73-83
16228363
Nucleic Acids Res. 2009 Jan;37(Database issue):D205-10
18984618
Hum Genet. 1981;58(4):425-8
7327566
Plant J. 2006 Nov;48(4):499-510
17059408
J Biol Chem. 2007 Dec 21;282(51):36914-22
17977822
Protein Expr Purif. 2007 May;53(1):24-30
17275326
Plant Physiol. 2006 Nov;142(3):911-22
17028153
EMBO J. 2004 Apr 21;23(8):1720-8
15057273
Microbiology (Reading). 2002 Aug;148(Pt 8):2273-2282
12177321
Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7540-5
11404477
Plant Physiol. 2000 Jan;122(1):75-84
10631251
J Biol Chem. 2008 Apr 4;283(14):9002-11
18230620
J Bacteriol. 1996 Apr;178(7):1946-54
8606169
Biochemistry. 2009 Jul 28;48(29):6705-11
19583219
Nat Struct Biol. 2001 Feb;8(2):156-60
11175906
Physiol Plant. 2002 May;115(1):9-24
12010463
Genetics. 1966 Jul;54(1):61-76
5961488
Gene. 1996 Dec 5;182(1-2):169-75
8982084
J Biotechnol. 2000 Feb 17;77(2-3):219-34
10682281
Biochem J. 1989 May 15;260(1):231-5
2775186
Mol Biol Evol. 2005 Dec;22(12):2343-53
16093570
Plant Physiol. 2001 Jan;125(1):50-3
11154294