Substrate specificity of nonribosomal peptide synthetase modules responsible for the biosynthesis of the oligopeptide moiety of cephabacin in Lysobacter lactamgenus (original) (raw)
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Insights into Cephamycin Biosynthesis: the Crystal Structure of CmcI from Streptomyces clavuligerus
Journal of Molecular Biology, 2006
Cephamycin C-producing microorganisms use two enzymes to convert cephalosporins to their 7a-methoxy derivatives. Here we report the X-ray structure of one of these enzymes, CmcI, from Streptomyces clavuligerus. The polypeptide chain of the enzyme folds into a C-terminal Rossmann domain and a smaller N-terminal domain, and the molecule packs as a hexamer in the crystal. The Rossmann domain binds S-adenosyl-L-methionine (SAM) and the demethylated product, S-adenosyl-L-homocysteine, in a fashion similar to the common binding mode of this cofactor in SAM-dependent methyltransferases. There is a magnesium-binding site in the vicinity of the SAM site with a bound magnesium ion ligated by residues Asp160, Glu186 and Asp187. The expected cephalosporin binding site near the magnesium ion is occupied by polyethyleneglycol (PEG) from the crystallisation medium. The geometry of the SAM and the magnesium binding sites is similar to that found in cathechol O-methyltransferase. The results suggest CmcI is a methyltransferase, and its most likely function is to catalyse the transfer of a methyl group from SAM to the 7a-hydroxy cephalosporin in the second catalytic reaction of cephamycin formation. Based on the docking of the putative substrate, 7a-hydroxy-Ocarbamoyldeacetylcephalosporin C, to the structure of the ternary CmcI-Mg 2C -SAM complex, we propose a model for substrate binding and catalysis. In this model, the 7-hydroxy group of the b-lactam ring ligates the Mg 2C with its a-side facing the methyl group of SAM at a distance that would allow methylation of the hydroxyl-group.
Biochemistry, 1995
Two clinically-important beta-lactam antibiotics, cephalothin and cefotaxime, have been observed by X-ray crystallography bound to the reactive Ser62 of the D-alanyl-D-alanine carboxypeptidase/transpeptidase of Streptomyces sp. R61. Refinement of the two crystal structures produced R factors for 3 sigma (F) data of 0.166 (to 1.8 A) and 0.170 (to 2.0 A) for the cephalothin and cefotaxime complexes, respectively. In each complex, a water molecule is within 3.1 and 3.6 A of the acylated beta-lactam carbonyl carbon atom, but is poorly activated by active site residues for nucleophilic attack and deacylation. This apparent lack of good stereochemistry for facile hydrolysis is in accord with the long half-lives of cephalosporin intermediates in solution (20-40 h) and the efficacy of these beta-lactams as inhibitors of bacterial cell wall synthesis. Different hydrogen binding patterns of the two cephalosporins to Thr301 are consistent with the low cefotaxime affinity of an altered penicillin-binding protein, PBP-2x, reported in cefotaxime-resistant strains of Streptococcus pneumoniae, and with the ability of mutant class A beta-lactamases to hydrolyze third-generation cephalosporins.
Advances in Biochemical Engineering/Biotechnology, 2004
Cephalosporin is one of the best b-lactam antibiotics, widely used in the treatment of infectious diseases. It is synthesized by Acremonium chrysogenum. The levels of cephalosporin produced by the improved strains obtained by classical mutation and selection procedures are still low compared to the penicillin titers obtained from the high-producing Penicillium chrysogenum strains. Most of the genes encoding the cephalosporin biosynthesis enzymes have been cloned, and some improvement of cephalosporin production has been achieved by removing bottlenecks in the pathway. One of the poorly-known steps involved in cephalosporin biosynthesis is the conversion of isopenicillin N into penicillin N catalyzed by the isopenicillin N epimerase system. This epimerization reaction is catalyzed by a two-component protein system encoded by the cefD1 and cefD2 genes that correspond, respectively, to an isopenicillinyl-CoA ligase and an isopenicillinyl-CoA epimerase. Comparative analysis of those proteins with others in the databanks provide evidence indicating that they are related to enzymes catalyzing the catabolism of toxic metabolites in animals. There are several biochemical mechanisms, reviewed in this article, for the biosynthesis of D-amino acids in secondary metabolites. The conversion of isopenicillin N to penicillin N in cephamycin-producing bacteria is mediated by a classical pyridoxal phosphate-dependent epimerase that is clearly different from the epimerization system existing in Acremonium chrysogenum. Modification of gene expression by directed manipulation of the cefD1-cefD2 bidirectional promoter region is a promising strategy for improving cephalosporin production. Improving our knowledge of the mechanism of epimerization systems is important if we wish to understand how microorganisms synthesize the high number of rare D-amino acids that are responsible, to a large extent, for the biological activities of many different secondary metabolites.
Journal of Biological Chemistry, 1996
Cephamycin C-producing microorganisms contain a two-protein enzyme system that converts cephalosporins to 7-methoxycephalosporins. Interaction between the two component proteins P 7 (M r 27,000) and P 8 (M r 32,000) has been studied by immunoaffinity chromatography using anti-P 7 and anti-P 8 antibodies, cross-linking with glutaraldehyde, and fluorescence spectroscopy analysis. Co-renaturation of the P 7 and P 8 polypeptides resulted in the formation of a protein complex with a molecular mass of 59 kDa, which corresponds to a heterodimer of P 7 and P 8 . Glutaraldehyde cross-linking of the polypeptides after assembly of the protein complex showed the presence of a single heterodimer form that reacted with antibodies against P 7 and P 8 . Each separate protein did not associate with itself into multimers. The P 7 ⅐P 8 complex co-purified by immunoaffinity chromatography from extracts of Nocardia lactamdurans and Streptomyces clavuligerus, suggesting that both proteins are present as an aggregate in vivo. Fluorescence spectroscopy studies of 5-methylaminonaphthalene-1-sulfonyl-P 7 in response to increasing concentrations of P 8 showed a blue shift in the fluorophore emission, indicating a conformational change of P 7 in response to the interaction of P 8 with an apparent dissociation constant of 47 M. NADH showed affinity for the P 7 component. The P 7 ⅐P 8 complex interacted strongly with the substrates S-adenosylmethionine and cephalosporin C, differently from that occurring with the separate P 7 or P 8 components, resulting in a strong blue shift in the fluorescence emission spectra of the complex.
Applied and Environmental Microbiology - AEM, 2005
Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastro- intestinal disease caused by Bacillus cereus. Due to its chemical structure, (D-O-Leu-D-Ala-L-O-Val-L-Val)3, cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules. The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide- deficient mutants. The valine-acti...
The 2.0 Å Crystal Structure of Cephalosporin Acylase
Structure, 2000
acid (7-ACA), a starting compound for industrial production Yeungnam University that can be obtained by chemical deacylation of CPC. The Dae-Dong, Kyungsan 712-749 total worldwide market value for cephalosporin antibiotics was Korea 6.9billionoutof6.9 billion out of 6.9billionoutof253 billion for the total pharmaceutical sales ‡ School of Food Biotechnology in 1998, and cephalosporin ranked fifth among the leading Woosong University therapeutic agents worldwide [1]. In the pharmaceutical indus-Daejon, 300-100 try, CPC is produced by bulk fermentation in the same manner Korea as penicillin G. The latter compound is subsequently converted § Department of Biological Structure and to 6-aminopenicillanic acid (6-APA) by penicillin G acylase Department of Biochemistry and (PGA), which is then used to synthesize penicillin antibiotics. Biomolecular Structure Center and In contrast, the production of 7-ACA is carried out in industry by Howard Hughes Medical Institute chemical methods using toxic compounds such as iminoether, University of Washington nitrosyl chloride, and methanol [2]. These chemical methods Seattle, Washington 98195 include several expensive steps and require thorough treatment of chemical wastes to overcome environmental safety problems. Therefore, an enzymatic conversion of CPC to 7-ACA Summary is of great interest in cephalosporin antibiotics manufacturing [3, 4]. The biggest problem preventing enzymatic industrial Background: Semisynthetic cephalosporins are primarily synproduction is that cephalosporin acylase (CA) takes glutarylthesized from 7-aminocephalosporanic acid (7-ACA), which is 7ACA (GL-7ACA) as a primary substrate and has a too low usually obtained by chemical deacylation of cephalosporin C substrate specificity for CPC to be applicable for industrial (CPC). The chemical production of 7-ACA includes, however, production of 7-ACA [5, 6, 7, 8] (Table 1). several expensive steps and requires thorough treatment of CAs have been categorized in two different ways. The first chemical wastes. Therefore, an enzymatic conversion of CPC one depends on whether a CA carries noticeable activity with to 7-ACA by cephalosporin acylase is of great interest. The respect to CPC. This leads to the "CPC acylases" with some biggest obstacle preventing this in industrial production is that activity on CPC, and the "GL-7ACA acylases," which hardly cephalosporin acylase uses glutaryl-7ACA as a primary subshow enzymatic activity toward CPC. Second, CAs have been strate and has low substrate specificity for CPC. grouped into five classes (CA I-CA V) on the basis of their gene structure, molecular mass and enzymatic properties [9]. These Results: We have solved the first crystal structure of a cephabiochemical properties are very similar within each class [10]. losporin acylase from Pseudomonas diminuta at 2.0 Å resolu-All CAs in the five classes have a broad spectrum of substrate tion. The overall structure looks like a bowl with two "knobs" specificity and are able to convert diverse substrates and subconsisting of helix-and strand-rich regions, respectively. The strate analogs (Table 1), but their activities on CPC vary from active site is mostly formed by the distinctive structural motif 0% to 4.0% relative to GL-7ACA. Out of five different classes of the N-terminal (Ntn) hydrolase superfamily. Superposition of CAs, class III CAs have the highest activity relative to CPC of the 61 residue active-site pocket onto that of penicillin G [5, 6, 7, 8]. acylase shows an rmsd in C␣ positions of 1.38 Å. This indicates The gene structure of the open reading frame of CAs varies structural similarity in the active site between these two enwith each enzyme but generally consists of a signal peptide zymes, but their overall structures are elsewhere quite different. followed by the ␣-subunit, a spacer sequence, then the -subunit. The genes of CAs are translated into an inactive single precur-Conclusion: The substrate binding pocket of the P. diminuta sor peptide that is posttranslationally modified into active encephalosporin acylase provides detailed insight into the ten key zymes with one ␣and one -subunit [11]. It is believed that the residues responsible for the specificity of the cephalosporin C posttranslational modification takes place by an autocatalytic side chain in four classes of cephalosporin acylases, and it process and that the resulting N-terminal residue (serine or thereby forms a basis for the design of an enzyme with an threonine) of the  chain plays a key role both as a nucleophile improved conversion rate of CPC to 7-ACA. The structure also in enzymatic catalysis and during the autocatalytic activation provides structural evidence that four of the five different step. As described by Li et al. [9], these modifications are a classes of cephalosporin acylases can be grouped into one general feature of the Ntn hydrolase superfamily [12, 13]. On family of the Ntn hydrolase superfamily. the other hand, the N-terminal nucleophile aminohydrolase (Ntn hydrolase) superfamily was defined by SCOP [14] as con