Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader

Abstract

Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

“…Sequence analysis revealed a putative AraC family transcriptional-regulator gene located upstream of the CYP153 gene cluster in both Actinobacteria and Proteobacteria, such as in Dietzia (15,18), Rhodococcus (27), Mycobacterium (28), Amycolicicoccus (29), Acinetobacter (13), and Alcanivorax (17,23,30). These findings implied a link between AraC family regulators (AFRs) and CYP153 proteins in bacterial alkane degradation, as reported previously (29).…”

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C 2 to C 4 Alkanes and Alkenes

“…By analogy with methanotrophs, NBB3 and NBB4 may switch the Smo and Hmo enzymes based on copper availability (21). There are problems with this hypothesis, however; smoX was transcribed in copper-containing medium (23), growth of NBB4 on C 2 to C 4 alkanes was almost entirely inhibited in copper-free medium or in the presence of allylthourea (ATU), a copper chelator (15), and the Smo gene cluster lacks an mmoD homolog, which is involved in copper regulation (21). Alternatively, Smo/Hmo may be a high-affinity/low-affinity pair.…”

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

“…There is an interesting parallel here with the dual-MMO methanotrophs. We have previously demonstrated that growth of NBB4 on ethene is associated with the SDIMO enzyme EtnABCD, as is the case in other ethenotrophic bacteria (Mattes et al, 2010;Coleman et al, 2011). The observation that HmoCAB is also capable of ethene oxidation indicates that this bacterium has distinct enzymes potentially capable of initiating growth on ethene.…”