Comparison of the 1.85 A structure of CYP154A1 from Streptomyces coelicolor A3(2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways

The genus Streptomyces produces two-thirds of microbially derived antibiotics. Polyketides form the largest and most diverse group of these natural products. Antibiotic diversity of polyketides is generated during their biosynthesis by several means, including postpolyketide modification performed by oxidoreductases, a broad group of enzymes including cytochrome P450 monooxygenases (CYPs). CYPs catalyze site-specific oxidation of macrolide antibiotic precursors significantly affecting antibiotic activity. Efficient manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of monooxygenases with activities toward a diverse array of chemical substrates. To begin to link structure to function of CYPs involved in secondary metabolic pathways of industrially important species, we determined the X-ray structure of Streptomyces coelicolor A3(2) CYP154A1 at 1.85 A and analyzed it in the context of the closely related CYP154C1 and more distant CYPs from polyketide synthase (EryF) and nonribosomal peptide synthetase (OxyB) biosynthetic pathways. In contrast to CYP154C1, CYP154A1 reveals an active site inaccessible from the molecular surface, and an absence of catalytic activities observed for CYP154C1. Systematic variations in the amino acid patterns and length of the surface HI loop correlate with degree of rotation of the F and G helices relative to the active site in CYP154A1-related CYPs, presumably regulating the degree of active site accessibility and its dimensions. Heme in CYP154A1 is in a 180 degrees flipped orientation compared with most other structurally determined CYPs.     

Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β-sheet on top of a long and a short α-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k _cat of 1.1 × 10² sec⁻¹. The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.     

Structure-guided activity restoration of the silkworm glutathione transferase Omega GSTO3-3

Glutathione transferases (GSTs) are ubiquitous detoxification enzymes that conjugate hydrophobic xenobiotics with reduced glutathione. The silkworm Bombyx mori encodes four isoforms of GST Omega (GSTO), featured with a catalytic cysteine, except that bmGSTO3-3 has an asparagine substitution of this catalytic residue. Here, we determined the 2.20-Å crystal structure of bmGSTO3-3, which shares a typical GST overall structure. However, the extended C-terminal segment that exists in all the four bmGSTOs occupies the G-site of bmGSTO3-3 and makes it unworkable, as shown by the activity assays. Upon mutation of Asn29 to Cys and truncation of the C-terminal segment, the in vitro GST activity of bmGSTO3-3 could be restored. These findings provided structural insights into the activity regulation of GSTOs.     

定点突变技术研究苏云金杆菌Cry1类晶体蛋白结构与功能的进展

近年来利用定点突变技术研究苏云金杆菌（Bacillus thuringiesis,Bt）杀虫晶体蛋白（Insecticidal crystal proteins,ICP）作用机制已取得良好进展.杀虫晶体蛋白不同结构域上氨基酸残基的突变将影响其稳定性,与受体的结合,不可逆的昆虫中肠膜插入及离子通道活性的强弱等.突变研究表明,结构域Ⅰ参与不可逆结合及插入昆虫中肠膜过程;结构域Ⅱ参与受体结合,包括初始结合与不可逆结合;结构域Ⅲ在杀虫特异性和维持三维结构的稳定性方面起重要作用,同时,可能参与离子通道的形成,受体结合和插入昆虫中肠膜过程.利用各种定点突变技术对各位点进行突变可以研究单一位点的功能,到目前为止,已有很多关于这方面的研究,并且筛选到了毒力提高的工程菌株.     

An improved method for culturing Streptomyces sahachiroi: biosynthetic origin of the enol fragment of azinomycin B

Azinomycin B is an environmental DNA crosslinking agent produced by the soil microorganism Streptomyces sahachiroi. While the agent displays potent cytotoxic activities against leukemic cell lines and animal mouse models, the lack of a consistent supply of the natural product has hampered detailed biological investigations on the compound, including its mode of action and biosynthesis. We report here a significant methodological improvement in the culturing of the bacterium, which allows reliable and steady production of the natural product in good yields. The key experimental step involves the culturing of the strain on dehydrated plates, followed by the generation of a two-stage starter culture and subsequent fermentation of the strain under nutrient-starved conditions. We illustrate use of this culture system by investigating the formation of the enol fragment of the molecule in isotopic labeling experiments with threonine and several advanced precursors (β-ketoamino acid 3, β-hydroxyamino aldehyde 4, and β-ketoaminoaldehyde 5). The results unequivocally show that threonine is the most advanced precursor accepted by the NRPS (non-ribosomal peptidyl synthetase) machinery for final processing and construction of the enol moiety of the natural product.     

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway

N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340 nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.     

Structure and mechanism of action of a cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus with broad specificity phosphatase activity

The crystal structure of Bacillus stearothermophilus PhoE (originally termed YhfR), a broad specificity monomeric phosphatase with a molecular mass of approximately 24 kDa, has been solved at 2.3 A resolution in order to investigate its structure and function. PhoE, already identified as a homolog of a cofactor-dependent phosphoglycerate mutase, shares with the latter an alpha/beta/alpha sandwich structure spanning, as a structural excursion, a smaller subdomain composed of two alpha-helices and one short beta-strand. The active site contains residues from both the alpha/beta/alpha sandwich and the sub-domain. With the exception of the hydrophilic catalytic machinery conserved throughout the cofactor-dependent phosphoglycerate mutase family, the active-site cleft is strikingly hydrophobic. Docking studies with two diverse, favored substrates show that 3-phosphoglycerate may bind to the catalytic core, while alpha-napthylphosphate binding also involves the hydrophobic portion of the active-site cleft. Combining a highly favorable phospho group binding site common to these substrate binding modes and data from related enzymes, a catalytic mechanism can be proposed that involves formation of a phosphohistidine intermediate on His10 and likely acid-base behavior of Glu83. Other structural factors contributing to the broad substrate specificity of PhoE can be identified. The dynamic independence of the subdomain may enable the active-site cleft to accommodate substrates of different sizes, although similar motions are present in simulations of cofactor-dependent phosphoglycerate mutases, perhaps favoring a more general functional role. A significant number of entries in protein sequence databases, particularly from unfinished microbial genomes, are more similar to PhoE than to cofactor-dependent phosphoglycerate mutases or to fructose-2,6-bisphosphatases. This PhoE structure will therefore serve as a valuable basis for inference of structural and functional characteristics of these proteins.     

Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707

Maltohexaose-producing amylase (G6-amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short-chain amylose (DP=17). Our previous crystallographic study showed that G6-amylase has nine subsites, from -6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6-amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6-amylase complexes with G6 and maltopentaose (G5). In the active site of the G6-amylase/G5 complex, G5 is bound to subsites -6 to -2, while G1 and G6 are found at subsites +2 and -7 to -2, respectively, in the G6-amylase/G6 complex. In both structures, the glucosyl residue located at subsite -6 is stacked to the indole moiety of Trp140 within a distance of 4A. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short-chain amylose by W140L is lower than that by W140Y or wild-type enzyme. The face-to-face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite -6 and to govern product specificity for G6 production.     

Structure of the neutrophil-activating protein from Helicobacter pylori

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.     

Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e⁻/H⁺ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met⁵⁸, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e⁻/H⁺ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.     

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.     

Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica

Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.     

O-糖基化起始糖基转移酶的活性与结构研究

多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T） 是催化N-乙酰氨基半乳糖（GalNAc）结合到蛋白质Ser或Thr上的糖基转移酶,是黏蛋白型O-糖基化修饰的起始糖基转移酶。ppGalNAc-T是一个酶家族,表达产物均为Ⅱ型膜蛋白。虽然氨基酸序列高度同源,但各成员具有独特的底物特异性和动力学特征。因此,ppGalNAc-T的底物作用机制是O-糖基化研究领域中的关键课题。近年来,通过利用定点突变及晶体结构解析技术,ppGalNAc-T中与底物相互作用的重要氨基酸残基以及由这些残基所形成的对底物结合起关键作用的空间构象逐渐被揭示,为了解ppGalNAc-T酶家族的底物作用机制及其蛋白结构与催化活性间的关系提供了理论依据。     

Role of glutamate-52 in the mechanism of L-lactate dehydrogenase from Bacillus stearothermophilus

A single residue of the NAD(H)-dependent lactate dehydrogenase (LDH) from Bacillus stearothermophilus has been changed in order to decrease substrate inhibition. The conserved aspartic acid residue at position 52 was replaced by glutamate using site-directed mutagenesis. The effect on substrate inhibition was measured. In the glutamate-52 mutant substrate inhibition is decreased twofold.     

Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway

The enzyme 2,6-dihydroxypyridine-3-hydroxylase catalyzes the sixth step of the nicotine degradation pathway in Arthrobacter nicotinovorans. The enzyme was produced in Escherichia coli, purified and crystallized. The crystal structure was solved at 2.6 Å resolution, revealing a significant structural relationship with the family of FAD-dependent aromatic hydroxylases, but essentially no sequence homology. The structure was aligned with those of the established family members, showing that the FAD molecules are bound at virtually identical locations. The reported enzyme is a dimer like most other family members, but its dimerization contact differs from the others. The binding position of NAD(P)H to this enzyme family is not clear. Since the reported enzyme accepts only NADH for flavin reduction in contrast to the other established members using NADPH, we searched through the structural alignment and found an indication for the position of the 2′-phosphate of NADPH that is in general agreement with mutational studies on a related enzyme, but contradicts a crystal soaking experiment. Using a bound glycerol molecule and the known substrate positions of three related enzymes as a guide, the substrate 2,6-dihydroxypyridine was placed into the active center. The access to the binding site is discussed. The new active center geometry introduces constraints that render some reaction scenarios more likely than others. It suggests that flavin is reduced at its out-position and then drawn into its in-position, where it binds molecular oxygen. The geometry is consistent with the proposal that peroxy-flavin is protonated by the solvent to yield the electrophilic hydroperoxy-flavin. The substrate is activated by two buried histidines but there is no appropriate base to store the surplus proton of the hydroxylated carbon atom. The implications of this problem are discussed.     

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys¹³) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.     

Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3.

The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied. Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions. The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid. However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid. In addition, the kcat value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination. Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme. We propose the possible reaction mechanism of this multifunctional enzyme based on these findings.     

Structure and mechanism of HpcH: a metal ion dependent class II aldolase from the homoprotocatechuate degradation pathway of Escherichia coli

Microorganisms are adept at degrading chemically resistant aromatic compounds. One of the longest and most well characterized aromatic catabolic pathways is the 4-hydroxyphenylacetic acid degradation pathway of Escherichia coli. The final step involves the conversion of 4-hydroxy-2-oxo-heptane-1,7-dioate into pyruvate and succinic semialdehyde. This reaction is catalyzed by 4-hydroxy-2-oxo-heptane-1,7-dioate aldolase (HpcH), a member of the divalent metal ion dependent class II aldolase enzymes that have great biosynthetic potential. We have solved the crystal structure of HpcH in the apo form, and with magnesium and the substrate analogue oxamate bound, to 1.6 A and 2.0 A, respectively. Comparison with similar structures of the homologous 2-dehydro-3-deoxygalactarate aldolase, coupled with site-directed mutagenesis data, implicate histidine 45 and arginine 70 as key catalytic residues.     

Crystal Structure of AhpE from Mycobacterium tuberculosis, a 1-Cys peroxiredoxin

All living systems require protection against the damaging effects of reactive oxygen species. The genome of Mycobacterium tuberculosis, the cause of TB, encodes a number of peroxidases that are thought to be active against organic and inorganic peroxides, and are likely to play a key role in the ability of this organism to survive within the phagosomes of macrophages. The open reading frame Rv2238c in M.tuberculosis encodes a 153-residue protein AhpE, which is a peroxidase of the 1-Cys peroxiredoxin (Prx) family. The crystal structure of AhpE, determined at 1.87 A resolution (R(cryst)=0.179, R(free)=0.210), reveals a compact single-domain protein with a thioredoxin fold. AhpE forms both dimers and octamers; a tightly-associated dimer and a ring-like octamer, generated by crystallographic 4-fold symmetry. In this native structure, the active site Cys45 is in its oxidized, sulfenic acid (S-O-H) state. A second crystal form of AhpE, obtained after soaking in sodium bromide and refined at 1.90 A resolution (R(cryst)=0.242, R(free)=0.286), reveals the reduced structure. In this structure, a conformational change in an external loop, in two of the four molecules in the asymmetric unit, allows Arg116 to stabilise the Cys45 thiolate ion, and concomitantly closes a surface channel. This channel is identified as the likely binding site for a physiological reductant, and the conformational change is inferred to be important for the reaction cycle of AhpE.