Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl) succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O-2-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons

Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

Since the characterization of cytochrome c552 as a multiheme nitrite reductase, research on this enzyme has gained major interest. Today, it is known as pentaheme cytochrome c nitrite reductase (NrfA). Part of the NH4+ produced from NO2- is released as NH3 leading to nitrogen loss, similar to denitrification which generates NO, N2O, and N2. NH4+ can also be used for assimilatory purposes, thus NrfA contributes to nitrogen retention. It catalyses the six-electron reduction of NO2- to NH4+, hosting four His/His ligated c-type hemes for electron transfer and one structurally differentiated active site heme. Catalysis occurs at the distal side of a Fe(III) heme c proximally coordinated by lysine of a unique CXXCK motif (Sulfurospirillum deleyianum, Wolinella succinogenes) or, presumably, by the canonical histidine in Campylobacter jejeuni. Replacement of Lys by His in NrfA of W. succinogenes led to a significant loss of enzyme activity. NrfA forms homodimers as shown by high resolution X-ray crystallography, and there exist at least two distinct electron transfer systems to the enzyme. In γ-proteobacteria (Escherichia coli) NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a pentaheme electron carrier (NrfB), in δ- and ε-proteobacteria (S. deleyianum, W. succinogenes), the NrfA dimer interacts with a tetraheme cytochrome c (NrfH). Both form a membrane-associated respiratory complex on the extracellular side of the cytoplasmic membrane to optimize electron transfer efficiency. This minireview traces important steps in understanding the nature of pentaheme cytochrome c nitrite reductases, and discusses their structural and functional features.publishe

Microbial sulfite respiration.

Despite its reactivity and hence toxicity to living cells, sulfite is readily converted by various microorganisms using distinct assimilatory and dissimilatory metabolic routes. In respiratory pathways, sulfite either serves as a primary electron donor or terminal electron acceptor (yielding sulfate or sulfide, respectively), and its conversion drives electron transport chains that are coupled to chemiosmotic ATP synthesis. Notably, such processes are also seen to play a general role in sulfite detoxification, which is assumed to have an evolutionary ancient origin. The diversity of sulfite conversion is reflected by the fact that the range of microbial sulfite-converting enzymes displays different cofactors such as siroheme, heme c, or molybdopterin. This chapter aims to summarize the current knowledge of microbial sulfite metabolism and focuses on sulfite catabolism. The structure and function of sulfite-converting enzymes and the emerging picture of the modular architecture of the corresponding respiratory/detoxifying electron transport chains is emphasized

Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme

A [3Cu:2S] cluster provides insight into the assembly and function of the Cu_Z site of nitrous oxide reductase

Nitrous oxide reductase (N2OR) is the only known enzyme reducing environmentally critical nitrous oxide (N2O) to dinitrogen (N2) as the final step of bacterial denitrification. The assembly process of its unique catalytic [4Cu:2S] cluster CuZ remains scarcely understood. Here we report on a mutagenesis study of all seven histidine ligands coordinating this copper center, followed by spectroscopic and structural characterization and based on an established, functional expression system for Pseudomonas stutzeri N2OR in Escherichia coli. While no copper ion was found in the CuZ binding site of variants H129A, H130A, H178A, H326A, H433A and H494A, the H382A variant carried a catalytically inactive [3Cu:2S] center, in which one sulfur ligand, SZ2, had relocated to form a weak hydrogen bond to the sidechain of the nearby lysine residue K454. This link provides sufficient stability to avoid the loss of the sulfide anion. The UV-vis spectra of this cluster are strikingly similar to those of the active enzyme, implying that the flexibility of SZ2 may have been observed before, but not recognized. The sulfide shift changes the metal coordination in CuZ and is thus of high mechanistic interest.publishe

Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase

Cytochrome cd(1) nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have been studied and found to be dominated by pronounced interactions between the c and the d(1) hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime example of intraprotein control of the electron-transfer rates by allosteric interactions