Cryogenic and Laser Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven Enzyme Protochlorophyllide Oxidoreductase

The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of protochlorophyllide (Pchlide), a key step in chlorophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex.The light-driven enzyme protochlorophyllide oxidoreductase (POR)³ (EC 1.3.1.33) catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide) (see Fig. 1A) (1). This reaction is a key step in the synthesis of chlorophyll and leads to profound changes in the morphological development of photosynthetic organisms through modification and reorganization of plastid membranes (2, 3). In addition to POR, nonflowering land plants, algae, and cyanobacteria possess a light-independent Pchlide reductase, which consists of three separate subunits and allows these organisms to produce chlorophyllide in the dark (4). Together with DNA photolyase (5), POR is one of only two enzymes studied so far that exhibit a direct, natural requirement for light and because mixing strategies are no longer required to initiate the reaction, it is possible to trigger catalysis on very fast time scales and at cryogenic temperatures. Consequently, POR has proven to be an excellent model system for studying the role of protein dynamics in driving enzyme catalysis (1).Open in a separate windowFIGURE 1.The light-driven reduction of Pchlide. A, the trans addition of hydrogen across the C-17–C-18 double bond of Pchlide to form chlorophyllide (Chlide) in the chlorophyll biosynthesis pathway is catalyzed by the light-driven enzyme POR. B, shown is a three-dimensional model of the POR-catalyzed reaction based on the structural homology model of POR (26) and the proposed mechanism of hydride and proton transfer (8). Upon activation by light, a hydride is transferred to the C-17 position of Pchlide from the pro-S face of NADPH (shown in yellow), and the proton at the C-18 position is derived from Tyr-193 (shown in cyan). The conserved Lys-197 residue (shown in magenta) is proposed to decrease the pK_a of the Tyr to facilitate the proton transfer reaction.In the POR catalytic cycle, a ternary enzyme-NADPH-Pchlide complex is formed. Following light activation of this complex, a hydride ion is transferred from the pro-S face of NADPH to the C-17 atom of Pchlide (6, 7). The valence of the C-18 atom is satisfied by proton transfer, which is suggested to originate from an active site tyrosine residue (8). The catalytic cycle of POR has been analyzed through the trapping of intermediates at cryogenic temperatures. Following the initial light-driven reaction (9), there are a series of subsequent (slower) dark reactions (10, 11). The light-driven step involves hydride transfer from NADPH to form a charge transfer complex, which then facilitates protonation of the pigment intermediate during the first of the “dark” reactions (12). Moreover, through laser activation of catalysis, we have shown that both of these H-transfer reactions proceed by quantum mechanical tunneling coupled to motions in the enzyme-substrate complex on the submicrosecond time scale (13). The final dark steps in the reaction cycle involve a series of ordered product release and cofactor binding steps linked to conformational changes in the enzyme (10, 11, 14). Ultrafast measurements have uncovered spectral changes on the picosecond timescale that are likely to represent conformational changes prior to Pchlide reduction (15–19). Previous excitation of POR with a laser pulse leads to a more efficient conformation of the active site and an enhancement in the catalytic efficiency of the enzyme (18).POR is a member of a large family of enzymes known as short chain dehydrogenases/reductases (SDR). These are single domain NAD(P)⁺- or NAD(P)H-binding oxidoreductases that exist generally as dimers or tetramers (20). A number of SDR enzymes (e.g. carbonyl reductase, alcohol dehydrogenase, and dihydrofolate reductase) have been good model systems for studying the dynamics linked to enzyme catalysis (21–23). This family of enzymes has been amenable to studies of biological H-tunneling (24–26), and in particular the unique light-activated properties of POR make it an excellent system for studying mechanisms of H-transfer and dynamics in this family of enzymes.Structures of several SDR family members are available, and these have enabled the construction of a homology model of POR from Synechocystis (27). This model comprises a central parallel β-sheet of seven β-strands, surrounded by nine α-helices, with an additional unique 33-residue insertion between the fifth and sixth β-sheets. The NADPH cofactor binds within the N-terminal region of the enzyme, which contains a common nucleotide-binding motif with a tight βαβ fold, termed the Rossmann fold (27). Importantly, a Tyr and a Lys residue are both absolutely conserved throughout all members of the SDR family and are critical for catalysis in a number of enzymes (28–31). A common mechanism has been proposed for this group of enzymes, involving a Tyr-X-X-X-Lys motif. The Lys residue in this motif is presumed to facilitate proton donation from the Tyr hydroxyl group to substrate through favorable perturbation of the hydroxyl group pK_a (8, 31). In POR, multiple turnover assays have also indicated that these Tyr and Lys residues are important for activity (8, 32, 33), leading to a proposed mechanism that involves proton transfer from the conserved Tyr residue to the C-18 position of Pchlide (8) (Fig. 1B). The close proximity of the Lys residue is thought to allow the deprotonation step to occur at physiological pH by lowering the apparent pK_a of the phenolic group of the Tyr (8). However, confirmation of the exact role of these conserved residues has been compromised by the limited levels of activity observed in previous studies of the variant enzymes (8, 32, 33), and a detailed evaluation of the role of the active site Tyr and Lys residues on the chemical steps (i.e. hydride and proton transfer) has not been reported. We address this deficiency in the current work by analyzing a number of site-specific mutant forms altered at Tyr-193 and Lys-197 in a thermophilic POR from Thermosynechococcus elongatus BP-1. This was achieved using steady-state (multiple turnover) and laser photoexcitation (single turnover) methods and by trapping transient reaction intermediates by fluorescence spectroscopy performed at cryogenic temperatures.     

Mechanistic Reappraisal of Early Stage Photochemistry in the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

The light-driven enzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This reaction is a key step in the biosynthesis of chlorophyll. Ultrafast photochemical processes within the Pchlide molecule are required for catalysis and previous studies have suggested that a short-lived excited-state species, known as I675*, is the first catalytic intermediate in the reaction and is essential for capturing excitation energy to drive subsequent hydride and proton transfers. The chemical nature of the I675* excited state species and its role in catalysis are not known. Here, we report time-resolved pump-probe spectroscopy measurements to study the involvement of the I675* intermediate in POR photochemistry. We show that I675* is not unique to the POR-catalyzed photoreduction of Pchlide as it is also formed in the absence of the POR enzyme. The I675* species is only produced in samples that contain both Pchlide substrate and Chlide product and its formation is dependent on the pump excitation wavelength. The rate of formation and the quantum yield is maximized in 50∶50 mixtures of the two pigments (Pchlide and Chlide) and is caused by direct energy transfer between Pchlide and neighboring Chlide molecules, which is inhibited in the polar solvent methanol. Consequently, we have re-evaluated the mechanism for early stage photochemistry in the light-driven reduction of Pchlide and propose that I675* represents an excited state species formed in Pchlide-Chlide dimers, possibly an excimer. Contrary to previous reports, we conclude that this excited state species has no direct mechanistic relevance to the POR-catalyzed reduction of Pchlide.     

Crystal Structure of the Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase Catalytic Complex (ChlN/ChlB)2

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL₂ bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)₂ complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)₂ complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel β-sheet surrounded by α-helices. The (ChlN/ChlB)₂ crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)₂(ChlL₂)₂ complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.     

Mercury Inhibits the Activity of the NADPH:Protochlorophyllide Oxidoreductase (POR)

The effect of Hg⁺⁺ was studied on the arrangement and photoactivity of NADPH:protochlorophyllide oxidoreductase (POR) in homogenates of dark-grown wheat (Triticum aestivum L.) leaves. 77 K fluorescence emission spectra of the homogenates were recorded before and after the irradiation of the homogenates and the spectra were deconvoluted into Gaussian components. The mercury treatment caused a precipitation of the membrane particles, which was followed by a remarkable decrease of the fluorescence yield. 10^-3 M Hg⁺⁺ decreased the ratio of the 655 nm-emitting protochlorophyllide (Pchlide) form to the 633 nm-emitting form. 10^-2 M Hg⁺⁺ shifted the short wavelength band to 629–630 nm and a 655 nm form was observed which was inactive on irradiation. This inhibition may be caused by serious alteration of the enzyme structure resulting in the trans-localisation of NADPH within the active site of POR.     

Site of Synthesis of NADPH: Protochlorophyllide Oxidoreductase in Rye (Secale cereale)

The site of synthesis of the plastid membrane-located enzyme, protochlorophyllide reductase, has been determined. Plastid ribosome-deficient and normal rye (Secale cereale L., cv Rheidol) plants were grown in darkness at 33°C and 22°C, respectively. Extracts from these plants were analyzed for the levels of different ribosomal RNAs and cytochrome f and the activity of a number of enzymes with well-established sites of synthesis. The results confirmed that the higher temperature had induced a specific inhibition of protein synthesis in the plastids. The activity and level of protochlorophyllide reductase was unaffected by growth at the higher temperature, suggesting it to be a cytoplasmically synthesized enzyme.     

Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

Photoregulation of Protochlorophyllide Oxidoreductase and Rubisco Large Subunit Accumulation in Phytochrome A-Deficient Transgenic Tobacco Plants

Some morphogenetic responses, induced by far red (FR) light in tobacco plants (Nicotiana tabacum L.), were studied. The inhibitory effect of FR irradiation on chlorophyll synthesis in transgenic plants with reduced phytochrome A content was almost absent. Phytochrome A-mediated repression of the por gene was demonstrated with the use of polyclonal antiserum against protochlorophyllide oxidoreductase. Continuous FR light induced the accumulation of Rubisco large subunits in wild-type but not in transgenic tobacco plants. Our data confirm the suggestion that phytochrome A mediates photoregulation of the synthesis of these proteins.     

Expression and Deletion Mutagenesis of Tryptophan Hydroxylase Fusion Proteins: Delineation of the Enzyme Catalytic Core

Abstract: cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1–98) or catalytic (amino acids 99–444) domains of the enzyme, were cloned and expressed as glutathione S -transferase fusion proteins in E. coli . The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S -transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity ( V _max) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99–444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.     

Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1.     

Formate Oxidase,an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family,Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

Formate oxidase of Aspergillus oryzae RIB40 contains an 8-replaced FAD with molecular mass of 799 as cofactor. The ¹H-NMR spectrum of the cofactor fraction obtained from the enzyme indicated that the 8-replaced FAD in the fraction was 8-formyl-FAD, present in open form and hemiacetal form. The oxidation-reduction potentials of the open and hemiacetal forms were estimated by cyclic voltammetry to be ?47 and ?177 mV vs. Normal Hydrogen Electrode respectively. The structure of the enzyme was constructed using diffraction data to 2.24 Å resolution collected from a crystal of the enzyme. His₅₁₁ and Arg₅₅₄ were situated close to the pyrimidine part of the isoalloxazine ring of 8-formyl-FAD in open form. The enzyme had 8-formyl-FAD, the oxidation potential of which was approximately 160 mV more positive than that of FAD, and the His-Arg pair at the catalytic site, unlike the other enzymes belonging to the glucose-methanol-choline oxidoreductase family.     

Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a β-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.     

工业真菌高效产酶突变技术与高产机制

工业真菌是酶制剂的重要生产微生物,其菌种改良具有重要应用价值。综述了产酶工业真菌的应用、突变育种技术进展和高效产酶机制的分析策略,尤其是基于组学水平的基因组、转录组和蛋白质组的分析等,最后总结了高产机制在菌株改良上的应用。     

Measurement and Identification of NADPH:Protochlorophyllide Oxidoreductase Solubilized with Triton X-100 from Etioplast Membranes of Squash Cotyledons

Etioplast membranes were solubilized with 1 mM Triton X-100in the presence of excess NADPH and protochlorophyllide to isolateNADPH:protochlorophyllide oxidoreductase. The activity of thisreductase was assayed as the formation of chlorophyllide bya single flash and was equivalent to the amount of photoactiveprotochlorophyllide-NADPH-enzyme complex present before illumination.The rate of regeneration of the phtoactive complex was estimatedfrom the time course of chlorophyllide formation under a longflash. The highest rate was 651 nmol chlorophyllide formed min^–1mg^–1 protein. Photoconversion of protochlorophyllide to chlorophyllide andregeneration of the photoactive protochlorophyllide-NADPH-enzymecomplex were not much affected in a pH range from 6 to 8, atleast for several minutes. The apparent dissociation constantsof the photoactive complex were 0.039 µM for protochlorophyllideand 0.44 µM for NADPH. Triton-solubilized etioplast membraneswere fractionated by glycerol density gradient centrifugationto isolate the NADPH:protochlorophyllide oxidoreductase. Mostof the 36,000-dalton protein, the major protein of the prolamellarbody was recovered in the fraction enriched by NADPH:protochlorophyllideoxidoreductase and protochlorophyllide. Protochlorophyll andcarotenoids were present in different fractions. This is evidencethat the 36,000-dalton protein has the activity of NADPH:protochlorophyllideoxidoreductase and specifically binds protochlorophyllide. Themost highly purified fraction of the enzyme showed an activityof 7.8 nmol chiorophyllide formed flash^–1 mg^–1 proteinand bound 11.1 nmol protochlorophyllide mg^–1 of protein. (Received April 28, 1982; Accepted June 29, 1982)     

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.     

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.     

产纤溶酶少根根霉菌株的诱变筛选

目的:通过对自南方小酒药中筛选得到的1株产纤溶酶的少根根霉Or株的诱变筛选,提高原有菌株的产酶能力。方法:以Or为出发菌株,进行亚硝基胍、紫外线、Co60诱变,以血纤维蛋白平板法为检测方法,筛选高产酶突变株。结果:经诱变传代后得到高产突变株8B,其产酶活性稳定为291.05U/mL,为原菌株产酶活力的6.34倍。该菌在血琼脂平板上不产生溶圈,诱变后孢子成熟提前8h。结论:物理诱变和化学诱变交替应用,能显著提高少根根霉Or株单位体积发酵液的产酶量,并能缩短孢子的成熟周期。该菌不具有溶血性,与已有报道的产纤溶酶的菌株不同。     

Site-directed Mutagenesis of the Catalytic Residues of Bovine Pancreatic Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterised endonuclease which cleaves double-stranded DNA to yield 5′ phosphory lated polynucleotides. Co-crystal structures of DNase I with two different oligonucleotides have revealed the presence of several residues (R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. The roles that these amino acids play in the catalytic mechanism have been investigated using site-directed mutagenesis. The following variants were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics of all six mutants with both DNA and a small chromophoric substrate, thymidine-3′,5′-di(p- nitrophenyl)-phosphate, were studied. Only R9A and E78T showed any significant turnover of the two substrates. D168S, H134Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3′,5′-di-n(p-nitrophenyl)-phosphate. These results demonstrate that H134, D168, D212 and H252 play a critical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provided general acid and general base catalysis. DNase I also requires Mg^{2 +}and E39 has been identified as a ligand for this metal ion. We propose that D168 serves as a ligand for a second Mg^{2 +}, and thus DNase I, uses a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mutagenesis results, structural information, homologies between DNase I from different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalytic role to each amino acid/metal ion.     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.