“…From studies of Escherichia coli cells expressing B. subtilis ctaA or Bacillus stearothermophilus ctaA, it has been demonstrated that the CtaA protein catalyzes heme A synthesis from heme O (3,20,26). Recently, Hegg and collaborators, using the B. subtilis ctaA gene expressed in E. coli, showed that heme A synthase activity is dependent on the presence of molecular oxygen and presented evidence that hydroxymethyl heme O (heme I) is a reaction intermediate (3).…”
Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed an affinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.
“…From studies of Escherichia coli cells expressing B. subtilis ctaA or Bacillus stearothermophilus ctaA, it has been demonstrated that the CtaA protein catalyzes heme A synthesis from heme O (3,20,26). Recently, Hegg and collaborators, using the B. subtilis ctaA gene expressed in E. coli, showed that heme A synthase activity is dependent on the presence of molecular oxygen and presented evidence that hydroxymethyl heme O (heme I) is a reaction intermediate (3).…”
Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed an affinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.
“…This observation led to speculation that HAS oxidizes heme O utilizing a P450-like mechanism, giving the unique situation of a heme cofactor acting on a heme substrate. However, there is currently no direct evidence to support these hypotheses (26,28).…”
mentioning
confidence: 99%
“…This observation led to speculation that HAS oxidizes heme O utilizing a P450-like mechanism, giving the unique situation of a heme cofactor acting on a heme substrate. However, there is currently no direct evidence to support these hypotheses ( , ). 1 Transformation of Heme B to Heme A Catalyzed by the Enzymes Heme O Synthase and Heme A Synthase a a In B. subtilis , HOS is denoted CtaB, while HAS is denoted CtaA.…”
Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase. Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde. We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase. Because E. coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes. We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D. We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase. Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.
“…Those enzymes have not been discovered that catalyze the processes in the biosynthesis of coenzyme F 430 from dihydrosirohydrochlorin, though the insertion of nickel to the porphyrin nucleus is known to occur at a relatively earlier stage of the biosynthesis and the rearrangement of side chains occurs after the insertion of nickel (Thauer and Bonacker, 1994). Circled numbers: 1, Glutamate-tRNA ligase + glutamate-tRNA reductase + glutamate-1-semialdehyde 2,1-aminomutase; 2, porphobilinogen synthase (ALA dehydrogenase); 3, hydroxymethylbilane synthase (PBG deaminase) + uroporphyrinogen III synthase (cosynthase); 4, uroporphyrinogen decarboxylase; 5, coproporphyrinogen oxidase; 6, protoporphyrinogen oxidase; 7, ferrochelatase; 8, holocytochrome c synthase (cytochrome c heme-lyase); 9, protoheme IX farnesyl transferase (Saiki et al, 1992); 10, oxidation of 8-CH 3 of heme O (Svensson et al, 1993;Sakamoto et al, 1999); 11, (magnesium-) chelatase (Castelfranco et al, 1994); 12, magnesium-protoporphyrin IX O-methyltransferase + magnesiumprotoporphyrin IX monomethylester (oxidative) cyclase + protochlorophyllide (4-vinyl) reductase (requiring NADP + and light) (Castelfranco et al, 1994); 13, (zinc-) chelatase (zinc-chlorophyll was discovered by Wakao et al, 1996); 14, uroporphyrinogen III C-methyl transferase (requiring S-adenosyl-l-methionine) (Warren et al, 1994); 15, Battersby, 1994; 16, dehydrogenase (NAD + / NADP + ) (Warren et al, 1994); 17, Mucha et al, 1985;Thauer and Bonacker, 1994). Circled numbers: 1, Glutamate-tRNA ligase + glutamate-tRNA reductase + glutamate-1-semialdehyde 2,1-aminomutase; 2, porphobilinogen synthase (ALA dehydrogenase); 3, hydroxymethylbilane synthase (PBG deaminase) + uroporphyrinogen III synthase (cosynthase); 4, uroporphyrinogen decarboxylase; 5, coproporphyrinogen oxidase; 6, protoporphyrinogen oxidase; 7, ferrochelatase; 8, holocytochrome c synthase (cytochrome c heme-lyase); 9, protoheme IX farnesyl transferase (Saiki et al, 1992); 10, oxidation of 8-CH 3 of heme O (Svensson et al, 1993;Sakamoto et al, 1999); 11, (magnesium-) chelatase (Castelfranco et al, 1994); 12, magnesium-protoporphyrin IX O-methyltransferase + magnesiumprotoporphyrin IX monomethylester (oxidative) cyclase + protochlorophyllide (4-vinyl) reductase (requiring NADP + and light) (Castelfranco et al, 1994); 13, (zinc-) chelatase (zinc-chlorophyll was discovered by Wakao et al, 1996); 14, uroporphyrinogen III C-methyl transferase (requiring S-adenosyl-l-methionine) (Warren et al, 1994); 15, Battersby, 1994; 16, dehydrogenase (NAD + /...…”
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