Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.     

Kinetics and comparative reactivity of human class I and class IIb histone deacetylases

Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.     

Characteristics of a new enantioselective thermostable dipeptidase from Brevibacillus borstelensis BCS-1 and its application to synthesis of a D-amino-acid-containing dipeptide

A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the D-Glu auxotroph Escherichia coli WM335 on a plate containing D-Ala-D-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M(r) of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P(1) and P(1)' site of Ala-Ala revealed that the ratio of the specificity constant (k(cat)/K(m)) for L-enantioselectivity to the P(1) site of Ala-Ala was 23.4 +/- 2.2 [E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(D,D)], while the D-enantioselectivity to the P(1)' site of Ala-Ala was 16.4 +/- 0.5 [E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(L,L)] at 55 degrees C. The enzyme was stable up to 55 degrees C, and the optimal pH and temperature were 8.5 and 65 degrees C, respectively. The enzyme was able to hydrolyze L-Asp-D-Ala, L-Asp-D-AlaOMe, Z-D-Ala-D-AlaOBzl, and Z-L-Asp-D-AlaOBzl, yet it could not hydrolyze D-Ala-L-Asp, D-Ala-L-Ala, D-AlaNH(2), and L-AlaNH(2.) The enzyme also exhibited beta-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-L-Asp-D-AlaOBzl.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.     

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Characterization of d-lactate dehydrogenase from Pediococcus acidilactici that converts phenylpyruvic acid into phenyllactic acid

The gene coding for D-lactate dehydrogenase (D-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The K(m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) for PPA were 2.9 mM, 305 s(-1), and 105 mM(-1) s(-1), respectively.     

The inhibition specificity of recombinant Penicillium funiculosum xylanase B towards wheat proteinaceous inhibitors

The filamentous fungus Penicillium funiculosum produces a mixture of modular and non-modular xylanases belonging to different glycoside hydrolase (GH) families. In the present study, we heterologously expressed the cDNA encoding GH11 xylanase B (XYNB) and studied the enzymatic properties of the recombinant enzyme. Expression in Escherichia coli led to the partial purification of a glutathione fusion protein from the soluble fraction whereas the recombinant protein produced in Pichia pastoris was successfully purified using a one-step chromatography. Despite O-glycosylation heterogeneity, the purified enzyme efficiently degraded low viscosity xylan [K(m)=40+/-3 g l(-1), V(max)=16.1+/-0.8 micromol xylose min(-1) and k(cat)=5405+/-150 s(-1) at pH 4.2 and 45 degrees C] and medium viscosity xylan [K(m)=34.5+/-3.2 g l(-1), V(max)=14.9+/-1.0 micromol xylose min(-1)k(cat)=4966+/-333 s(-1) at pH 4.2 and 45 degrees C]. XYNB was further tested for its ability to interact with wheat xylanase inhibitors. The xylanase activity of XYNB produced in P. pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a K(i) of 89.7+/-8.5 and 2.9+/-0.3 nM, respectively, whereas no inhibition was detected with TAXI-II. Physical interaction of both TAXI-I and XIP-I with XYNB was observed using titration curves across a pH range 3-9.     

A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

Cloning, expression and characterisation of Erwinia carotovora L-asparaginase

Bacterial L-asparaginases (E.C. 3.5.1.1) have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukaemia. L-asparaginase from Erwinia carotovora NCYC 1526 (ErA) was cloned and expressed in E. coli. The enzyme was purified to homogeneity by a two-step procedure comprising cation-exchange chromatography and affinity chromatography on immobilised L-asparagine. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. Molecular modelling studies were also employed to create a model of ErA, based on the known structure of the Erwinia chrysanthemi enzyme. The molecular model was used to help interpret biochemical data concerning substrate specificity and catalytic mechanism of the enzyme. The kinetic parameters of selected substrates were determined at various pH values, and the pH-dependence profiles of V(max) and V(max)/K(m) were analyzed. The pH-dependence of V(max) shows one transition in the acidic pH range with pK(a)=5.4, and the pH-dependence of V(max)/K(m) exhibits two transitions with pK(a)=5.4 and 8.5. Based on analysis of alternative substrates and molecular modelling studies, it was concluded that the pK(a) at the acidic pH range corresponds to the active site residues Asp115 or Glu82, whereas the pK(a) observed at the alkaline pH range is not due to substrate amino group ionisation, but rather is the result of enzyme ionisation. The effect of temperature and viscosity on the catalytic activity of the enzyme was also investigated and it was concluded that the rate-limiting step of the catalytic reaction is relevant to structural transitions of the protein. Thermodynamic analysis of the activity data showed that the activation energies are dependent on the substrate, and entropy changes appear to be the main determinant contributing to substrate specificity.     

Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.     

Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in k(cat). The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction. The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.     

Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS-PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70-80 degrees C (k(cat)/K(m)=8.3 mM(-1) s(-1)). In comparison with that of P. furiosus CP (k(cat)/K(m)=667 mM(-1) s(-1)), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.     

Galactose mutarotase: pH dependence of enzymatic mutarotation

Here we report pH dependence of kinetic parameters for the mutarotation of alpha-D-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli. The values of k(cat) and k(cat)/K(m) for the mutarotation of alpha-D-galactose were found to be 1.84 x 10(4) s(-1) and 4.6 x 10(6) M(-1) s(-1), respectively, at pH 7.0 and 27 degrees C. The corresponding values for alpha-D-glucose were 1.9 x 10(4) s(-1) and 5.0 x 10(5) M(-1) s(-1). Inasmuch as the value of k(cat)/K(m) for the reaction of alpha-D-galactose is 10 times that for alpha-D-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of alpha-D-glucose should not be diffusion controlled. Therefore, pH-rate profiles should not be distorted by diffusion. The k(cat) for the mutarotation of alpha-D-glucose is independent of pH. Therefore, either the enzyme-substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion. The profile of log k(cat)/K(m) versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and -2 on the alkaline side. The values of pK(a) are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated. On the acid side, ring opening of alpha-D-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate. A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms. In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation. The groups are postulated to be His 104, His 175, and Glu 309. Mutations of these residues grossly impair catalytic activity. Variants H104Q- and E309Q-GalM display sufficient activity to allow profiles of log k(cat)/K(m) versus pH to be constructed. Both profiles show breaks on the acid side corresponding to pK(a) values of 5.8 for H104Q and 6.3 for E309Q. Apparently, ring opening of alpha-D-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants.     

Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation.

A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 degrees C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 degrees C compared to that at 20 degrees C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 degrees C with a k(cat)/K(m) value of 0.87 micro m(-1) x s(-1). When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T(1) refolding assay at 10 and 20 degrees C, the protein exhibited higher activity at 10 degrees C with a k(cat)/K(m) value of 0.50 micro m(-1) x s(-1). These k(cat)/K(m) values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.     

Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli

To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K(m) values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k(cat) values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k(cat)/K(m)). The K(m) and k(cat) values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-beta-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.     

Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.     

Purification and some characteristics of a recombinant dimeric rhizobium meliloti beta-galactosidase expressed in escherichia coli

A recombinant Rhizobium meliloti beta-galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl beta-D-galactopyranoside (PNPG) and o-nitrophenyl beta-D-galactopyranoside (ONPG) with K(m)(PNPG) and K(m)(ONPG) of 1 mM at 25 degrees C. The k(cat)/K(m) ratios for both substrates were approximately 70 mM(-1) sec(-1), indicating no clear preference for either PNPG or ONPG, unlike E. coli beta-galactosidase. After non-denaturing electrophoresis, active beta-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl beta-D-galactopyranoside (BNG) and diazo blue B.     

A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.