Search

Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-l-phenylethanol. The subsequent carboxylation of acetophenone to benzoylacetate is catalyzed by an acetophenone-induced enzyme, which has been purified and studied. The same enzyme is involved in acetophenone metabolism in the absence of ethylbenzene. Acetophenone carboxylase consists of five subunits with molecular masses of 70, 15, 87, 75, and 34 kDa, whose genes (apcABCDE) form an apparent operon. The enzyme is synthesized at high levels in cells grown on ethylbenzene or acetophenone, but not in cells grown on benzoate. During purification, acetophenone carboxylase dissociates into inactive subcomplexes consisting of the 70-, 15-, 87-, and 75-kDa subunits (apcABCD gene products) and the 34-kDa subunit (apcE gene product), respectively. Acetophenone carboxylase activity was restored by mixing the purified subcomplexes. The enzyme contains I [Zn.sup.2+] ion per [alpha][beta][gamma][delta] core complex and is dependent on the presence of [Mg.sup.2+] or [Mg.sup.2+]. In spite of the presence of Zn in the enzyme, it is strongly inhibited by [Zn.sup.2+] ions. Carboxylation of acetophenone is dependent on ATP hydrolysis to ADP and [P.sub.i], exhibiting a stoichiometry of 2 mol ATP per mol acetophenone carboxylated. The enzyme shows uncoupled ATPase activity with either bicarbonate or acetophenone in the absence of the second substrate. These observations indicate that both substrates may be phosphorylated, which is consistent with isotope exchange activity observed with deuterated acetophenone and inhibition by carbamoylphosphate, a structural analogue of carboxyphosphate. A potential mechanism of ATP-dependent acetophenone carboxylation is suggested. doi: 10.1128/JB.01423-09

We show that translation initiation factor EF-Tu plays a second important role in cell shape maintenance in the bacterium Bacillus subtilis. EF-Tu localizes in a helical pattern underneath the cell membrane and colocalizes with MreB, an actin-like cytoskeletal element setting up rod cell shape. The localization of MreB and of EF-Tu is interdependent, but in contrast to the dynamic MreB filaments, EF-Tu structures are more static and may serve as tracks for MreB filaments. In agreement with this idea, EF-Tu and MreB interact in vivo and in vitro. Lowering of the EF-Tu levels had a minor effect on translation but a strong effect on cell shape and on the localization of MreB, and blocking of the function of EF-Tu in translation did not interfere with the localization of MreB, showing that, directly or indirectly, EF-Tu affects the cytoskeletal MreB structure and thus serves two important functions in a bacterium. Bacillus subtilis | bacterial cytoskeleton | cell shape www.pnas.org/cgi/doi/10.1073/pnas.0911979107

A new method is described by using an amide ligation reaction for the characterization of acyl carrier protein (ACP)-bound reaction intermediates. This novel method has characterized the ACP-mediated biosynthesis of the unique 2,3-epoxyhexanoyl moiety of calcium-dependent antibiotic (CDA), which has disclosed a new type of FAD-dependent oxidase HxcO with enoyl-ACP epoxidase activity, as well as a second enoyl-ACP epoxidase, HcmO.

A method to selectively alter the heterocyclic metal binding subunit of Bacitracin A by the synthesis of heterocyclic building blocks that were successfully coupled to the linear decapeptide and subsequently cyclized using the excised bacitracin PCP-TE bidomain is described. The generated bacitracin derivatives showed bacterial activity, indicating the possibility of altering the biological important heterocyclic subunit and overcoming existing limitations for the application of bacitracin.

The identification of a gene cluster for mannosylerythritol lipid (MEL) biosynthesis, which consists of five open reading frames, is described. The mutation analysis demonstrates that putative acyltransferases Mac1 and Mac2 are essential for MEL biosynthesis and acetyltransferase Mat1 catalyzes the acetylation of mannosylerythritol lipids in Ustilago maydis.

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin ([TEII.sub.srf]) and bacitracin ([TEII.sub.bac]). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For [TEII.sub.srf], a [K.sub.M] of 0.9 [micro]M and a [k.sub.cat] of 95 [min.sup.-1] was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with [TEII.sub.srf]. These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyI-CoAs as 4'PP donors. polyketide synthase | peptide antibiotic

Research has been conducted on the cyclic decapeptide antibiotic tyrocidine. The evaluation of the three- and four-domain versions of the tyrocidine synthetase B module has been carried out via the use of the rapid quench techniques in investigating the ability of the module's domain to epimerise aminoacyl thioester and dipeptidyl-S-enzyme and the details are discussed.

Research reveals that epimerization domains are functionally portable to A domains, which epimerize altered substrates to synthesize nonribosomal peptide synthetases. Data suggest that epimerization activity requires the presence of peptidyl carrier protein domain or T domain.

The role of the condensation domain in preventing misinitiation and timing of epimerization has been studied with focus on control of directionality in nonribosomal peptide synthesis. Epimerization domains of elongation modules were seen to be able to, in principle, epimerize aminoacyl S-Ppant and peptidyl-S-Ppant substrates, but efficiency for epimerizing the noncognate aminoacyl-S-Ppant substrates seems to be lowered. Natural peptides of pharmacological interest are often synthesized from bacteria/fungi by nonribosomal peptide synthetases.

The isolation and structural characterization of capistruin, a new lasso peptide from Burkholderia thailandensis E264, is described. Mass spectrometric and NMR spectroscopic studies have shown capistruin as a new representative of lasso peptides and the heterologous production of capistruin in Escherichia coli is established.

The biochemical proof is presented that the aldehyde intermediate, formed by the release of peptide from the protein template in an NAD(P)H-dependent reduction step catalyzed by the adjacent reductase, is further reduced by an aldoreductase, LgrE, in an NADPH-dependent manner to form the final product gramicidin A, N-formyl-pentadecapeptide-enthanolamine. To determine the potential use of the two reductases in the construction of hybrid NRPSs, their ability to accept a variety of different substrates in vitro is tested.

The recombinant cyclization domain of the streptomyces coelicolor calcium-dependent antibiotic (CDA) nonribosomal peptide synthetase (NRPS) is a versatile tool for the chemoenzymatic generation of daptomycin derivatives. The findings reveal that calcium-dependent antibiotic (CDA) cyclase is a versatile catalyst that can be used to generate novel daptomycin derivatives that are otherwise difficult to obtain by chemical modification of the parental tridecapeptide.

Results show that C-terminal E-domains of the nonribosomal peptide synthetases are involved in protein-protein recognition. Data reveal that C-terminal modules interacting in trans can be fused together in cis without the loss of activity. Furthermore, non-interacting modules in trans become active when they fuse together in cis.

A study was conducted to investigate the catalytic function of the putative formylation domain and the mode of action of N-formylation in linear gramicidin biosynthesis in order to test its interaction with the natural neighboring domains and the timing of the initiation reaction of the nonribosomal peptide synthetases (NRPS) assembly line. The experimental data indicate that the F domain is essential for the initiation of linear gramicidin biosynthesis.

Several biochemical, crystallographic and cellular studies are conducted to observe the direct and site-specific synthesis of thymine dimmer lesions, which is very important phenomenon to learn how DNA lesions induce mutations and how the lesions are removed from genome by the cells. The results show that the peptide nucleic acids (PNA) reagents are very useful in recognizing the targeted sequence in a large genomic DNA fragment, which can be further used to generate selectivity by the irradiation of the undesired lesion.

A new approach to obtain hybrid nonribosomal peptide synthetases (NRPSs) is used to show that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. It is concluded that the surgery in the linkers between entire modules seems to be a robust approach to rationally design NRPSs in nonribosomal peptide antibiotic producer strains.