Wael Ismail

Bacterial phenylalanine and phenylacetate catabolic pathway revealed

Aromatic compounds constitute the second most abundant class of organic substrates and environmental pollutants, a substantial part of which (e.g., phenylalanine or styrene) is metabolized by bacteria via phenylacetate. Surprisingly, the bacterial catabolism of phenylalanine and phenylacetate remained an unsolved problem. Although a phenylacetate metabolic gene cluster had been identified, the underlying biochemistry remained largely unknown. Here we elucidate the catabolic pathway functioning in 16% of all bacteria whose genome has been sequenced, including Escherichia coli and Pseudomonas putida. This strategy is exceptional in several aspects. Intermediates are processed as CoA thioesters, and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2-epoxide by a distinct multicomponent oxygenase. The reactive nonaromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin. This isomerization is followed by hydrolytic ring cleavage and β-oxidation steps, leading to acetyl-CoA and succinyl-CoA. This widespread paradigm differs significantly from the established chemistry of aerobic aromatic catabolism, thus widening our view of how organisms exploit such inert substrates. It provides insight into the natural remediation of man-made environmental contaminants such as styrene. Furthermore, this pathway occurs in various pathogens, where its reactive early intermediates may contribute to virulence.

Genome-scale comparison and constraint-based metabolic reconstruction of the facultative anaerobic Fe(III)-reducer Rhodoferax ferrireducens

BackgroundRhodoferax ferrireducens is a metabolically versatile, Fe(III)-reducing, subsurface microorganism that is likely to play an important role in the carbon and metal cycles in the subsurface. It also has the unique ability to convert sugars to electricity, oxidizing the sugars to carbon dioxide with quantitative electron transfer to graphite electrodes in microbial fuel cells. In order to expand our limited knowledge about R. ferrireducens, the complete genome sequence of this organism was further annotated and then the physiology of R. ferrireducens was investigated with a constraint-based, genome-scale in silico metabolic model and laboratory studies.ResultsThe iterative modeling and experimental approach unveiled exciting, previously unknown physiological features, including an expanded range of substrates that support growth, such as cellobiose and citrate, and provided additional insights into important features such as the stoichiometry of the electron transport chain and the ability to grow via fumarate dismutation. Further analysis explained why R. ferrireducens is unable to grow via photosynthesis or fermentation of sugars like other members of this genus and uncovered novel genes for benzoate metabolism. The genome also revealed that R. ferrireducens is well-adapted for growth in the subsurface because it appears to be capable of dealing with a number of environmental insults, including heavy metals, aromatic compounds, nutrient limitation and oxidative stress.ConclusionThis study demonstrates that combining genome-scale modeling with the annotation of a new genome sequence can guide experimental studies and accelerate the understanding of the physiology of under-studied yet environmentally relevant microorganisms.

Study of Anoxic and Oxic Cholesterol Metabolism by Sterolibacterium denitrificans

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1S(T). The second enzyme of the proposed pathway, cholest-4-en-3-one-Delta1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

Initial Steps in the Anoxic Metabolism of Cholesterol by the Denitrifying Sterolibacterium denitrificans

The anoxic metabolism of the ubiquitous triterpene cholesterol is challenging because of its complex chemical structure, low solubility in water, low number of active functional groups, and the presence of four alicyclic rings and two quaternary carbon atoms. Consequently, the aerobic metabolism depends on oxygenase catalyzed reactions requiring molecular oxygen as co-substrate. Sterolibacterium denitrificans is shown to metabolize cholesterol anoxically via the oxidation of ring A, followed by an oxygen-independent hydroxylation of the terminal C-25 of the side chain. The anaerobic hydroxylation of a tertiary carbon using water as oxygen donor is unprecedented and may be catalyzed by a novel molybdenum containing enzyme.

Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase.

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the beta-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP(+)-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.

Cholest-4-en-3-one-δ1-dehydrogenase: A flavoprotein catalyzing the second step in anoxic cholesterol metabolism

ABSTRACT The anoxic metabolism of cholesterol was studied in the denitrifying bacterium Sterolibacterium denitrificans, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ1-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ1-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ1-dehydrogenases from other organisms (highest amino acid sequence identity with that from Pseudoalteromonas haloplanktis), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.

A Novel Testosterone Catabolic Pathway in Bacteria

Forty years ago, Coulter and Talalay (A. W. Coulter and P. Talalay, J. Biol. Chem. 243:3238-3247, 1968) established the oxygenase-dependent pathway for the degradation of testosterone by aerobes. The oxic testosterone catabolic pathway involves several oxygen-dependent reactions and is not available for anaerobes. Since then, a variety of anaerobic bacteria have been described for the ability to degrade testosterone in the absence of oxygen. Here, a novel, oxygenase-independent testosterone catabolic pathway in such organisms is described. Steroidobacter denitrificans DSMZ18526 was shown to be capable of degrading testosterone in the absence of oxygen and was selected as the model organism in this study. In a previous investigation, we identified the initial intermediates involved in an anoxic testosterone catabolic pathway, most of which are identical to those of the oxic pathway demonstrated in Comamonas testosteroni. In this study, five additional intermediates of the anoxic pathway were identified. We demonstrated that subsequent steps of the anoxic pathway greatly differ from those of the established oxic pathway, which suggests that a novel pathway for testosterone catabolism is present. In the proposed anoxic pathway, a reduction reaction occurs at C-4 and C-5 of androsta-1,4-diene-3,17-dione, the last common intermediate of both the oxic and anoxic pathways. After that, a novel hydration reaction occurs and a hydroxyl group is thus introduced to the C-1α position of C(19)steroid substrates. To our knowledge, an enzymatic hydration reaction occurring at the A ring of steroid compounds has not been reported before.

Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans

The aerobic degradation of steroids by bacteria has been studied in some detail. In contrast, only little is known about the anaerobic steroid catabolism. Steroidobacter denitrificans can utilize testosterone under both oxic and anoxic conditions. By conducting metabolomic investigations, we demonstrated that S. denitrificans adopts the 9,10-seco-pathway to degrade testosterone under oxic conditions. This pathway depends on the use of oxygenases for oxygenolytic ring fission. Conversely, the detected degradation intermediates under anoxic conditions suggest a novel, oxygenase-independent testosterone catabolic pathway, the 2,3-seco-pathway, which differs significantly from the aerobic route. In this anaerobic pathway, testosterone is first transformed to 1-dehydrotestosterone, which is then reduced to produce 1-testosterone followed by water addition to the C-1/C-2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione. The A-ring of this compound is cleaved by hydrolysis as evidenced by H218O-incorporation experiments. Regardless of the growth conditions, testosterone is initially transformed to 1-dehydrotestosterone. This intermediate is a divergence point at which the downstream degradation pathway is governed by oxygen availability. Our results shed light into the previously unknown cleavage of the sterane ring structure without oxygen. We show that, under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring.

Structure and mechanism of the diiron benzoyl-coenzyme a epoxidase BOXB

The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the non-aromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoyl-CoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O2 must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester.

Initial steps in anoxic testosterone degradation by Steroidobacter denitrificans.

Steroid compounds have many important physiological activities in higher organisms. Testosterone and related steroids are important environmental contaminants that disrupt the endocrine systems of animals. The degradation of steroids, especially under anoxic conditions, is challenging because of their complex chemical structure. A denitrifying gamma-proteobacterium, Steroidobacter denitrificans, able to grow anaerobically on a variety of steroids as the sole carbon and energy source was adopted as a model organism to study the anoxic degradation of testosterone. We identified the initial intermediates involved in the anoxic testosterone degradation pathway of S. denitrificans. We demonstrated that under anoxic conditions, S. denitrificans initially oxidizes testosterone to 1-dehydrotestosterone, which is then transformed to androsta-1,4-diene-3,17-dione. In addition, it seems that androst-4-en-3,17-dione can also be directly produced from testosterone by S. denitrificans cells. In general, the initial steps of anoxic testosterone degradation by S. denitrificans are similar to those of the oxic pathway demonstrated in Comamonas testosteroni.