Dataset Information

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily.

ABSTRACT: The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH(4)) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH(4) and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure-function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C(2)-C(4) alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis-this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3?nmol?min(-1) per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C(2)-C(4) alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible.

SUBMITTER: Coleman NV

PROVIDER: S-EPMC3246247 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily.

Coleman Nicholas V NV Le Nga B NB Ly Mai A MA Ogawa Hitoha E HE McCarl Victoria V Wilson Neil L NL Holmes Andrew J AJ

The ISME journal 20110728 1

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH(4)) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH(4) and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure-function relationships i ...[more]

PMID: 21796219

Similar Datasets

Project description:The cytochrome P450 monooxygenases (CYP450, CYP, P450) catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways. Although CYP superfamily has been systematically studied in a few species, the genome-scale research about it in rice has not been done.In this study, a total of 355 CYPs encoded by 326 genes were identified in japonica genome. The OsCYP genes are classified into 10 clans including 45 families according to phylogenetic analysis. More than half of the genes are distributed in 53 tandem duplicated gene clusters. Intron-exon structure of OsCYPs exhibits highly conserved and specificity within a family, and divergences of duplicate genes in gene structure result in non-functionalization, neo-functionalization or sub-functionalization. Selection pressure analysis showed that rice CYPs are under purifying selection. The microarray data analysis shows that some genes are tissue-specific expression, such as OsCYP710A5 and OsCYP71X14 in endosperm, OsCYP99A3 and OsCYP78A16 in root and OsCYP93G2 and OsCYP97D7 in leaf. Analysis of RNA-seq data derived from rice leaf developmental gradient indicates that some OsCYPs exhibit zone-specific expression patterns. OsCYP87C2, OsCYP96B5, OsCYP96B8 and OsCYP84A5 were specifically expressed in leaf base and transitional zone. The transcripts of lineages II and IV-1 members were highly abundant in maturing zone. Eighty three OsCYPs are differentially expressed in response to drought stress, of which OsCYP51G3, OsCYP709C9, OsCYP709C5, OsCYP81A6, OsCYP72A18 and OsCYP704A5 are strongly induced and OsCYP78A16, OsCYP89C9 and OsCYP704A5 are down-regulated significantly, and some of the results were validated by qPCR. And 23 up-regulated and 17 down-regulated genes are specific to Osbhlh148 mutation under drought stress. Compared to those in wild type, the changes in transcript levels of several genes are slight in the mutant, such as OsCYP51G3, OsCYP94C2, OsCYP709C9 and OsCYP709C5.The whole-genomic analysis of rice P450 superfamily provides a clue to understanding biological function of OsCYPs in development regulation and drought stress response, and is helpful to rice molecular breeding.

Project description:Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, "Candidatus Nitrosocosmicus franklandus" C13 and "Candidatus Nitrosotalea sinensis" Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (?C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from "Ca Nitrosocosmicus franklandus" and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits "Ca Nitrosocosmicus franklandus" and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from "Ca Nitrosocosmicus franklandus," and it does not compete with NH3 for binding at the active site.IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

Dataset Information

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily.

Publications

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure