The ACE inhibitor captopril inhibits ACN-1 to control dauer formation and aging.

The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in Caenorhabditis elegans, Drosophila and rodents, but its mechanism is not well defined. Here, we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril-hypersensitive mutants. We identified a missense mutation that causes a partial loss of function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNA interference promoted dauer larvae formation, suggesting that acn-1 is a daf gene. Captopril-mediated lifespan extension was abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 influence lifespan by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control.

Structure and mechanism of the alkane-oxidizing enzyme AlkB.

Alkanes are the most energy-rich form of carbon and are widely dispersed in the environment. Their transformation by microbes represents a key step in the global carbon cycle. Alkane monooxygenase (AlkB), a membrane-spanning metalloenzyme, converts straight chain alkanes to alcohols in the first step of the microbially-mediated degradation of alkanes, thereby playing a critical role in the global cycling of carbon and the bioremediation of oil. AlkB biodiversity is attributed to its ability to oxidize alkanes of various chain lengths, while individual AlkBs target a relatively narrow range. Mechanisms of substrate selectivity and catalytic activity remain elusive. Here we report the cryo-EM structure of AlkB, which provides a distinct architecture for membrane enzymes. Our structure and functional studies reveal an unexpected diiron center configuration and identify molecular determinants for substrate selectivity. These findings provide insight into the catalytic mechanism of AlkB and shed light on its function in alkane-degrading microorganisms.

The ACE-inhibitor drug captopril inhibits ACN-1 to control dauer formation and aging.

The renin-angiotensin-aldosterone system (RAAS) plays a well-characterized role regulating blood pressure in mammals. Pharmacological and genetic manipulation of the RAAS has been shown to extend lifespan in C. elegans , Drosophila , and rodents, but its mechanism is not well defined. Here we investigate the angiotensin-converting enzyme (ACE) inhibitor drug captopril, which extends lifespan in worms and mice. To investigate the mechanism, we performed a forward genetic screen for captopril hypersensitive mutants. We identified a missense mutation that causes a partial loss-of-function of the daf-2 receptor tyrosine kinase gene, a powerful regulator of aging. The homologous mutation in the human insulin receptor causes Donohue syndrome, establishing these mutant worms as an invertebrate model of this disease. Captopril functions in C. elegans by inhibiting ACN-1, the worm homolog of ACE. Reducing the activity of acn-1 via captopril or RNAi promoted dauer larvae formation, suggesting acn-1 is a daf gene. Captopril-mediated lifespan extension xwas abrogated by daf-16(lf) and daf-12(lf) mutations. Our results indicate that captopril and acn-1 control aging by modulating dauer formation pathways. We speculate that this represents a conserved mechanism of lifespan control. Summary Statement: Captopril and acn-1 control aging. By demonstrating they regulate dauer formation and interact with daf genes, including a new DAF-2(A261V) mutant corresponding to a human disease variant, we clarified the mechanism.

An Overview of the Electron-Transfer Proteins That Activate Alkane Monooxygenase (AlkB).

Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron-sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research.

An alkane monooxygenase (AlkB) family in which all electron transfer partners are covalently bound to the oxygen-activating hydroxylase.

Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert CH bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.

Investigation of the prevalence and catalytic activity of rubredoxin-fused alkane monooxygenases (AlkBs).

Interest in understanding the environmental distribution of the alkane monooxygenase (AlkB) enzyme led to the identification of over 100 distinct alkane monooxygenase (AlkB) enzymes containing a covalently bound, or fused, rubredoxin. The rubredoxin-fused AlkB from Dietzia cinnamea was cloned as a full-length protein and as a truncated protein with the rubredoxin domain deleted. A point mutation (V91W) was introduced into the full-length protein, with the goal of assessing how steric bulk in the putative substrate channel might affect selectivity. Based on activity studies with alkane and alkene substrates, the rubredoxin-fused AlkB oxidizes a similar range of alkane substrates relative to its rubredoxin domain-deletion counterpart. Oxidation of terminal alkenes generated both an epoxide and a terminal aldehyde. The products of V91W-mutant-catalyzed oxidation of alkenes had a higher aldehyde-to-epoxide ratio than the products formed in the presence of the wild type protein. These results are consistent with this mutation causing a structural change impacting substrate positioning.