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.enoyl-CoA hydratase | epoxide | oxepin | oxygenase | phenylacetic acid T he biggest challenge for organisms using aromatic compounds as growth substrates is to overcome the stabilizing resonance energy of the aromatic ring system. This aromatic structure makes the substrates unreactive toward oxidation or reduction and thus requires elaborate degradation strategies. How microorganisms cope with this problem depends primarily on the availability of oxygen (1). Aerobic pathways use oxygen both for hydroxylation and for cleavage of the ring (2, 3). In contrast, under anaerobic conditions the common strategy consists of activation by CoA-thioester formation, shortening of the side chain, and energy-driven ring reduction, which also applies to phenylacetate catabolism (ref. 4 and literature cited therein).The aerobic strategy is illustrated by the metabolism of phenylacetate and phenylacetyl-CoA, which are derived from a variety of substrates such as phenylalanine, lignin-related phenylpropane units, 2-phenylethylamine, phenylalkanoic acids with an even number of carbon atoms, or even environmental contaminants such as styrene and ethylbenzene (5-7). Rarely, phenylalanine is hydroxylated to tyrosine, which can be converted into 4-hydroxyphenylpyruvate, followed by hydroxylation to homogentisate (2,5-dihydroxyphenylacetate) as the central intermediate. The aromatic ring of homogentisate then is split by a ring-cleaving homogentisate dioxygenase, and finally fumarate and acetoacetate are produced (8). In most cases, however, phenylalanine is converted into phenylacetate. A conventional aero...

SummaryToluene is anoxically degraded to CO 2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (␣), 90 (␣Ј), 12 (␤) and 10 kDa (␥) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the ␣-and ␣Ј-subunits were identical, suggesting a C-terminal degradation of half of the ␣-subunits to give the ␣Ј-subunit. The composition of native enzyme therefore appears to be ␣ 2 ␤ 2 ␥ 2 . A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the ␣Ј-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.

The mechanism of the electron transfer from the soluble protein plastocyanin to the multiprotein complex of photosystem I from spinach has been studied in detail. The two kinetic components of P700+ reduction by plastocyanin after a laser flash, showing a constant half-life of 11 microseconds and a variable half-life of the second-order reaction, respectively, are used to monitor the electron transfer from bound and soluble plastocyanin. The effect of increasing concentration of reduced plastocyanin on both of these kinetic components and the competition by oxidized plastocyanin is used to estimate the individual dissociation constants of the complex between the proteins in each of its oxidized and reduced state. The dissociation constant of oxidized plastocyanin is about six times larger than that of 7 microM found for reduced plastocyanin and purified PSI. Consistent with this result the midpoint redox potential of plastocyanin bound to photosystem I either in equilibrium with soluble plastocyanin or after cross-linking to photosystem I is found to be 50-60 mV higher than that of soluble plastocyanin. It is concluded that the driving force of the intracomplex electron transfer is decreased in favor of an optimized turnover of photosystem I. Double-flash excitation shows that oxidized plastocyanin has to leave the complex after the electron transfer before a new reduced plastocyanin molecule can bind to photosystem I. This release of oxidized plastocyanin with a half-life of about 60 microseconds limits the turnover of photosystem I. All data are consistently described by a model including the formation of a complex at a single binding site of photosystem I. Differences in the rate and binding constants are discussed with respect to the structure and the electrostatic and hydrophobic interactions stabilizing the complex as well as their modification by the membrane environment in situ.

The modular strategy of a template-assembled synthetic protein (TASP) was used for the de novo synthesis of a 122-residue, antiparallel four-helix bundle protein which accommodates two bis-histidine ligated heme groups. The cyclic decapeptide template contains four cysteine residues with different protecting groups which allow coupling of the unprotected helices carrying bromoacetyl units either at the N-terminus or the ε-amino group of a C-terminal lysine residue. The amphiphilic helices realize a water-soluble model of the cytochrome b core with two parallel heme-binding helices alternating with two antiparallel helices shielding the two hydrophobic heme binding sites. The spectral properties resemble those of the natural protein. Characterization by mass spectrometry and circular dichroism support the anticipated structure. The free energy of folding shows a stabilizing effect by the two heme groups which have respective redox midpoint potentials of −106 and −170 mV. This modular protein combines the advantage of the structural organization of a TASP with the incorporation of functional groups.

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site‐specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C‐terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid‐targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix‐assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.