Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.Many eubacteria are able to grow on linear alkanes by virtue of alkane hydroxylases (AHs) that activate alkanes to 1-alkanols. These are then further metabolized by alcohol and aldehyde dehydrogenases to fatty acids, which enter the central metabolism (34). AHs belong to several different oxygenase classes. Shortchain-length (C 2 to C 4 )-alkane degraders possess enzymes related to the soluble and particulate methane monooxygenases (34), while integral membrane nonheme iron AHs related to AlkB of Pseudomonas putida GPo1 were found in medium-chain-length (MCL) (C 5 to C 11 )-and especially in long-chain-length (LCL) (ՆC 12 )-alkane-degrading Alpha-, Beta-, and Gammaproteobacteria and high-GϩC gram-positive bacteria (34, 37).Several strains in our collection of alkane degraders grow well on MCL alkanes but could not be shown to contain AlkB homologs that act on MCL alkanes (some of these strains do contain AlkB homologs that hydroxylate LCL alkanes); these strains include the following. (i) Gordonia sp. strain 7E1C (Rhodococcus rhodochrous NCIMB 12566) is of interest because it is able to oxidize substituted phenoxy propane to phenoxy propanoic acids when pregrown on n-alkanes (17). It is known to contain an alkane-inducible cytochrome P450 (2).(ii) For Rhodococcus erythropolis NRRL B-16531 and Q15, none of the integral membrane AHs cloned from these isolates could be shown to act on MCL alkanes (39), even though these and other R. erythropolis isolates grow well on such alkanes (38). (iii) Of several hexane-degrading strains isolated from a trickling-bed bioreactor (32, 39), only 2 of 15 strains tested contained AlkB homologs that oxidize MCL alkanes (J. B. van Beilen et al., unpublished data). This left 13 strains for which growth on hexane initially could not be attributed to known enzyme systems. One of these strains, Sphingomonas sp. strain HXN-200, contains a soluble alkane hydroxylase, which is proposed to be responsible for a range of useful hydroxylation and epoxidation reactions of cyclic compounds such as pyrrolidines, pyrrolidinones, azetidines, azetidinones, piperidines, and piperidinones (4-6, 24, 25). Mycobacterium sp. strain HXN-1500, a strain able to convert limonene to perillyl alcohol (35), was found to contain a soluble alkane hydroxylase that is closely related to the Acinetobacter sp. strain EB104 hexane hydroxylase (CYP...
Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4⌬608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion. INTRODUCTIONRab GTPases serve as important regulators of membrane transport in eukaryotic cells (Deneka et al., 2003b). Most rabs are ubiquitously expressed, but some have a more restricted distribution. For instance, rab27a is highly expressed in melanocytes and hematopoietic and other secretory cells (Tolmachova et al., 2004). Mutations causing loss of rab27a function in human result in defects of pigmentation (Bahadoran et al., 2001) and defects in the granule exocytosis pathway in cytotoxic T lymphocytes (CTLs) (Menasche et al., 2000). This rare autosomal recessive disease is called Griscelli syndrome type II (GS2) (Sanal et al., 2002).Mutations in the genes encoding myosin-Va and the rab27a effector melanophilin also cause a pigmentation phenotype in human (Pastural et al., 1997;Menasche et al., 2003) and mice. These observations led to the identification of a ternary complex consisting of rab27a/melanophilin/myosin-Va that is essential for the normal function of melanosomes (Wu et al., 2002). A related complex containing rab27a/myRIP/myoVIIa seems to be important for melanosome localization in retinal pigment epithelium (Amraoui et al., 2002) and secretory granules in the PC12 pheochromocytoma cell line (Desnos et al., 2003).Melanocytes and hematopoietic cells combine the functions of lysosomes and secretory granules into a hybrid organelle, the melanosome and secretory lysosome, respectively. Secretory lysosomes are particularly found in cells of the hematopoietic lineage, such as natural killer cells, CTLs, mast cells, dendritic cells, B cells, and neutrophils. They have an acidic lumenal pH and contain lysosomal enzymes. Ne...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.