Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7

Abstract

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C 5 to C 30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C 5 ) to tetracosane (C 24 ) using NADH as a cofactor, with greatest activity on the C 15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe 2؉ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium.

Thermophilic bacteria are potential sources of novel Rieske non-heme iron oxygenases

“…In another study, the purified large subunit of a novel alkane monooxygenase (belonging to Class B ROs), identified from a cold-tolerant Pusillimonas sp. T7-7, showed NADH-dependent alkane monooxygenase activity (Li et al 2013). Transformation of aromatic hydrocarbons has also been attained by heterologous expression of the terminal oxygenase components using non-specific ET proteins complemented by the host strain (Mukerjee-Dhar et al 2005).…”

“…Interestingly, few bacterial RO homologs with novel functions, such as oxidative cyclization during biosynthesis of certain antibiotics, hydroxylation and desaturation of short-chain tertiary alcohols and alkane monooxygenation, have been reported in recent years (Sydor et al 2011; Schäfer et al 2012; Li et al 2013). This suggests that ROs bear much more catalytic potential than previously realized.…”

Characterization and application of a novel bioemulsifier in crude oil degradation by Acinetobacter beijerinckii ZRS

“…A. beijerinckii ZRS was isolated from an oil‐contaminated soil sample from the Xinjiang oil field in China and the culture conditions for this strain were based on Huang et al . T7‐7 was isolated from the seabed mud of the Bohai Sea in a petroleum‐contaminated region . Diesel oil was purchased from a local service station of Sinopec Group and the light crude oil was donated by Shengli Oil Field of China.…”

Survival and Energy Producing Strategies of Alkane Degraders Under Extreme Conditions and Their Biotechnological Potential

“…In addition, integral membrane non-heme iron (AlkB) or cytochrome P450 enzymes (CYP153) oxidize pentane (C 5 ) to hexadecane (C 16 ), and alkanes longer than heptadecane are metabolized by a novel alkane dioxygenase, putative flavin-binding monooxygenase (AlmA), or long-chain alkane monooxygenase (LadA) ( Maeng et al, 1996 ; Feng et al, 2007 ; Throne-Holst et al, 2007 ). However, a recent study showed that a novel Rieske-type alkane monooxygenase degrades pentane (C 5 ) to tetracosane (C 24 ) using NADH as a cofactor ( Li et al, 2013 ). In addition, many bacteria possess multiple alkane hydroxylases (AH), and therefore, it is possible that a wide range of alkanes can be degraded by one microorganism ( Whyte et al, 1998 ; Rozhkova-Novosad et al, 2007 ; Amouric et al, 2010 ; Liu et al, 2011 ; Park et al, 2017 ).…”