Conformational flexibility of the C terminus with implications for substrate binding and catalysis revealed in a new crystal form of deacetoxycephalosporin C synthase

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron-oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308-311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268-299) is rotated by about 16 degrees towards the active site. The last 12 residues (300-311) are disordered. Structures for various enzyme-substrate and enzyme-product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304-306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron.     

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Studies on the active site of deacetoxycephalosporin C synthase

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel filtration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A resolution and belonged to the R3 space group (unit cell dimensions: a=b=106.4 A, c=71.2 A; alpha=beta=90 degrees, gamma=120 degrees (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry defined by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A, three nitrogen or oxygen atoms at 2.11 A and one other light atom at 2.04 A. For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A, another O atom at 2.08 A and one at 2.03 A. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.     

高浓度底物下青霉素扩环酶的活性评价与底物抑制现象

本文对青霉素扩环酶(Penicillin expandase,也称Deacetoxycephalosporin C synthase,DAOCS)在高浓度青霉素G下的底物抑制现象进行初步评价与表征,筛选适合工业应用条件的高活力突变体。我们通过HPLC对已报道的几个DAOCS高活力突变体在青霉素G浓度5.6至500 mmol/L间的比活力进行定量测定,并与不同催化反应动力学模型的理论推测变化趋势比较,发现DAOCS野生型酶及高活力突变体H4、H5、H6与H7在高浓度青霉素G条件下均表现出明显的底物抑制现象,但是变化趋势不同。野生型酶与突变体H4的比活力先上升后下降,与竞争性抑制模型预测不符。突变体H5、H6与H7的比活力变化呈现更复杂的变化趋势。在所有测试的突变体中,H6的活性显著高于其他突变体酶。青霉素G对野生型DAOCS的底物抑制现象符合非竞争性抑制模型的预测。而部分突变体表现出复杂的底物抑制行为,表明其具有更复杂的作用机制。在高底物浓度下筛选具有较强催化活性的青霉素扩环酶突变体对于推动其在工业生产中的应用具有重要指导作用。     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

The structural basis of cephalosporin formation in a mononuclear ferrous enzyme

Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus. In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2. The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water. Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct. Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed. This species is stabilized by the negative charge of succinate on the iron. The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself. Requisite groups of penicillin are within 1 A of the expected position of a ferryl oxygen in the enzyme-penicillin complex.     

Alteration of the co-substrate selectivity of deacetoxycephalosporin C synthase. The role of arginine 258

Deacetoxycephalosporin C synthase is an iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the 5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine residue reduced activity to about 5% of the wild-type enzyme with 2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not co-substrates for the wild-type enzyme, were utilized by the R258Q mutant. These 2-oxoacids "rescued" catalytic activity to the level observed for the wild-type enzyme as judged by penicillin N and G conversion. These co-substrates underwent oxidative decarboxylation as observed for 2-oxoglutarate in the normal reaction with the wild-type enzyme. Crystal structures of the iron(II)- 2-oxo-3-methylbutanoate (1.5 A), and iron(II)-2-oxo-4-methylpentanoate (1.6 A) enzyme complexes were obtained, which reveal the molecular basis for this "chemical co-substrate rescue" and help to rationalize the co-substrate selectivity of 2-oxoglutaratedependent oxygenases.     

Contrasting fates for 6-alpha-methylpenicillin N upon oxidation by deacetoxycephalosporin C synthase (DAOCS) and deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS).

6-alpha-methylpenicillin N was synthesised via known routes from 6-aminopenicillanic acid, and tested as a substrate for recombinant DAOCS and DAOC/DACS. Incubation with DAOCS resulted in conversion of 2-oxoglutarate without oxidation of the penicillin substrate ('uncoupled turnover'). Incubation with DAOC/DACS resulted in oxidation to the cephem aldehyde. This is the first example of substrate-induced 'uncoupled turnover', which has been proposed to be an editing mechanism for these enzymes.     

New strategy of site-directed mutagenesis identifies new sites to improve <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> deacetoxycephalosporin C synthase activity toward penicillin G

Based on multiple sequence alignment of different deacetoxycephalosporin C synthase (DAOCSs) and the crystal structure of Streptomyces clavuligerus DAOCS, 2-oxoglutarate, and penicillin G triple complex, ten residues (Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213) not directly involved in substrate recognition were selected as mutational targets. Twenty one mutants were generated and characterized, and five (Q126M, T213V, S261M, S261A, and Y184A) showed improved activity toward penicillin G, with 1.45- to 4.50-fold increment in the k _cat/K _m. Q126, T213, and S261 are identified for the first time, as sites with significant effect on enzyme activity.     

Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k_cat/K_m ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k_cat/K_m values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k_cat/K_m ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the alpha-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.     

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

The biosynthesis of cephalosporins involving a thiozolidine ring expansion is catalyzed by deacetoxycephalosporin C synthase (DAOCS). In this study, three DAOCS isozymes were cloned and expressed as active enzymes together with Streptomyces jumonjinensis DAOCS that was newly isolated and partially characterized. The enzymes showed excellent substrate conversion for penicillin G, phenethicillin, ampicillin and carbenicillin, but they were less effective in the ring expansion of penicillin V, amoxicillin and metampicillin. Streptomyces clavuligerus DAOCS was the most active among the recombinant enzymes. The results also showed that truncation of 20 amino acids at the C-terminus of the Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase polypeptide did not affect penicillin ring expansion.     

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.

We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.     

Crystal structure of carbapenem synthase (CarC)

The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron- and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C2221), consistent with solution studies. CarC monomers contain a double-stranded beta-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with l-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis.     

Elucidation of active site residues of Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering flavonols

Arabidopsis thaliana flavonol synthase (aFLS) catalyzes the production of quercetin, which is known to possess multiple medicinal properties. aFLS is classified as a 2-oxoglutarate dependent dioxygenase as it requires ferrous iron and 2-oxoglutarate for catalysis. In this study, the putative residues for binding ferrous iron (H221, D223 and H277), 2-oxoglutarate (R287 and S289) and dihydroquercetin (H132, F134, K202, F293 and E295) were identified via computational analyses. To verify the proposed roles of the identified residues, 15 aFLS mutants were constructed and their activities were examined via a spectroscopic assay designed in this study. Mutations at H221, D223, H277 and R287 completely abolished enzymes activities, supporting their importance in binding ferrous iron and 2-oxoglutarate. However, mutations at the proposed substrate binding residues affected the enzyme catalysis differently such that the activities of K202 and F293 mutants drastically decreased to approximately 10% of the wild-type whereas the H132F mutant exhibited approximately 20% higher activity than the wild-type. Kinetic analyses established an improved substrate binding affinity in H132F mutant (Km: 0.027+/-0.0028 mM) compared to wild-type (Km: 0.059+/-0.0063 mM). These observations support the notion that aFLS can be selectively mutated to improve the catalytic activity of the enzyme for quercetin production.     

Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion

Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands.     

Structure of proline 3-hydroxylase. Evolution of the family of 2-oxoglutarate dependent oxygenases.

Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases catalyse oxidative reactions in a range of metabolic processes including the hydroxylation of proline and lysine residues during the post-translational modification of collagen. 2-OG oxygenases commonly require ascorbate for full activity. In the vitamin C deficient disease, scurvy, reduced activity of 2-OG oxygenases results in impaired formation of collagen. Here we report the crystal structure of bacterial proline 3-hydroxylase from Streptomyces sp., an enzyme which hydroxylates proline at position 3, the first of a 2-OG oxygenase catalysing oxidation of a free alpha-amino acid. Structures were obtained for the enzyme in the absence of iron (to 2.3A resolution, R=20.2%, Rfree=25.3%) and that complexed to iron (II) (to 2.4A resolution, R=19.8%, Rfree=22.6%). The structure contains conserved motifs present in other 2-OG oxygenases including a 'jelly roll' beta strand core and residues binding iron and 2-oxoglutarate, consistent with divergent evolution within the extended family. The structure differs significantly from many other 2-OG oxygenases in possessing a discrete C-terminal helical domain. Analysis of the structure suggests a model for proline binding and a mechanism for uncoupling of proline and 2-OG turnover.     

Oxygen derepresses deacetoxycephalosporin C synthase and increases the conversion of penicillin N to cephamycin C in Streptomyces clavuligerus.

When dissolved oxygen (DO) was maintained at saturation level during batch fermentations of Streptomyces clavuligerus (NRRL 3585), the accumulation of the intermediate penicillin N was lowered while formation of the end product cephamycin C was increased relative to fermentations without DO control. The specific activity of the penicillin ring-expansion enzyme deacetoxycephalosporin C synthase (DAOCS) was increased 2.3-fold under oxygen saturated conditions, whereas the penicillin ring-cyclizing enzyme isopenicillin N synthase (IPNS) showed only a 1.3-fold increase. Thus oxygen derepression of DAOCS appears to be an important regulatory mechanism in the conversion of penicillin N to cephamycin C in S. clavuligerus. IPNS, an early acting enzyme in cephamycin C biosynthesis, and DAOCS, which acts late in the pathway, both disappeared from cell extracts at 60 h, just prior to cessation of cephamycin production.     

Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin

Human alphaB-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide (54FLRAPSWF61) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alphaA-crystallin. To determine whether the positive charge on arginine 56 (R56) influences these functions, we prepared human alphaB-crystallin mutants in which R56 was deleted (DeltaR56) or replaced by alanine (R56A). To determine if the effects are specific to R56, we generated two additional mutant proteins in which the two neighboring amino acids were deleted (DeltaL55 and DeltaA57). Dynamic light scattering studies suggested that none of the mutations affected the oligomeric mass of the protein. Far-ultraviolet circular dichroism (UV CD) spectra revealed greater helicity in the secondary structures of R56A and DeltaR56 compared to that of the wild-type (Wt) protein. Near-UV CD spectra showed that the tertiary structure is perturbed in all mutants. Insulin and citrate synthase aggregation assays showed 38 and 30% improvement of chaperone function in DeltaR56 compared to that of the Wt. In contrast, the R56A mutant lost most of its chaperone function. Deletion mutants, DeltaL55 and DeltaA57, showed no significant changes in the chaperone function compared to that of the Wt. The DeltaR56 mutant had a higher surface hydrophobicity than the Wt, but the R56A mutant had a lower hydrophobicity. Our data show paradoxical effects of the deletion and substitution of R56 and imply that the chaperone function of human alphaB-crystallin is dictated not only by the positive charge on R56 but also by the conformational change that it bestows on the protein.     

Isopenicillin N synthase and desacetoxycephalosporin C synthase activities during defined medium fermentations of Streptomyces clavuligerus: effect of oxygen and iron supplements

When the level of dissolved oxygen was increased to saturation in defined media fermentations of Streptomyces clavuligerus, the total duration of activity of the penicillin ring cyclization enzyme, isopenicillin N synthase (IPNS), was extended by at least 20 h; however, no increase in the stability of the ring expansion enzyme, desacetoxycephalosporin C synthase (DAOCS), was observed. Consequently, the conversion of the excreted intermediate penicillin N to cephamycin C was 15-20% less efficient at this high oxygen concentration. The increased dissolved oxygen level also led to the complete loss of IPNS and DAOCS activities for 4 h during the period of fastest growth, and the rate of specific cephamycin C production fell to zero. A several hundred fold increase in the level of iron in the defined media resulted in a sixfold improvement in the rate of specific cephamycin C production after 60 h fermentation. This increased rate appeared to be due to an elevation in the in vivo activities of a number of the cephamycin biosynthetic enzymes, particularly those catalysing later pathway steps.