Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin

The side chain of the antifungal antibiotic ansatrienin A from Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC)-derived moiety. This moiety is also observed in trace amounts of omega-cyclohexyl fatty acids (typically less than 1% of total fatty acids) produced by S. collinus. Coenzyme A-activated CHC (CHC-CoA) is derived from shikimic acid through a reductive pathway involving a minimum of nine catalytic steps. Five putative CHC-CoA biosynthetic genes in the ansatrienin biosynthetic gene cluster of S. collinus have been identified. Plasmid-based heterologous expression of these five genes in Streptomyces avermitilis or Streptomyces lividans allows for production of significant amounts of omega-cyclohexyl fatty acids (as high as 49% of total fatty acids). In the absence of the plasmid these organisms are dependent on exogenously supplied CHC for omega-cyclohexyl fatty acid production. Doramectin is a commercial antiparasitic avermectin analog produced by fermenting a bkd mutant of S. avermitilis in the presence of CHC. Introduction of the S. collinus CHC-CoA biosynthetic gene cassette into this organism resulted in an engineered strain able to produce doramectin without CHC supplementation. The CHC-CoA biosynthetic gene cluster represents an important genetic tool for precursor-directed biosynthesis of doramectin and has potential for directed biosynthesis in other important polyketide-producing organisms.     

Production of doramectin by rational engineering of the avermectin biosynthetic pathway

In an attempt to construct a strain that produces doramectin, the loading module of Ave polyketide synthase (PKS) from Streptomyces avermitilis M1 was replaced with a cyclohexanecarboxylic (CHC) unique loading module from phoslactomycin PKS. Additionally, the CHC-CoA biosynthetic gene cassette was introduced into the engineered strain, which provided the precursor for directed biosynthesis of doramectin. The doramectin production ability of the final mutant S. avermitilis TG2002 was increased about six times and the ratio of Dor to Ave was enhanced 300 times more than the original strain.     

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.     

Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.     

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.     

The missing link in petrobactin biosynthesis: asbF encodes a (-)-3-dehydroshikimate dehydratase

The siderophore petrobactin harbors unique 3,4-dihydroxybenzoyl iron-liganding groups. These moieties are known to be synthesized from shikimate pathway precursors, but no reports of the biosynthetic enzymes responsible for this conversion have been published. The gene encoding AsbF from Bacillus thuringiensis 97-27 was overexpressed in an Escherichia coli host. AsbF rapidly and efficiently transforms (-)-3-dehydroshikimate (DHS) into 3,4-dihydroxybenzoate (k(cat)(DHS) = 217 +/- 10 min(-1); K(m)(DHS) = 125 +/- 14 microM) at 37 degrees C and has an absolute requirement for divalent metal. Finally, the pH versus k(cat)(DHS) profile revealed two ionizable groups (pK(a1) = 7.9 +/- 0.1, and pK(a2) = 9.3 +/- 0.1).     

A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus

Polyketide synthases cannot be functional unless their apo-acyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in approximately 93% reduction of FDM production, and complementation of the fdmW::aac (3)IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a K(m) of 5.8 microM and a k(cat)/K(m) of 8.1 microM(-1) x min(-1), to heterologous ACPs, such as the TcmM ACP with a K(m) of 1.0 x 10(2) microM and a k(cat) /K(m) of 0.6 microM(-1) x min(-1). These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.     

Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.     

Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum

The phosphoglucomutase gene from a wild type Fusarium oxysporum strain (F3), was homologously expressed, under the control of the constitutive promoter of gpdA of Aspergillus nidulans. The transformant produced elevated levels of phosphoglucomutase activity compared to the wild type, a fact that facilitated the subsequent purification procedure. The enzyme (FoPGM) was purified to homogeneity applying three anion exchange and one gel filtration chromatography steps. The native enzyme revealed a monomeric structure with a molecular mass of 60 kDa, while the isoelectric point was 3.5. FoPGM was active in pH ranged from 6.0 to 8.0, with an optimum using 3-(N-morpholino)propanesulfonic acid buffer at 7.0, while loss of activity was observed when phosphate buffer was used in the above mentioned pH range. The optimal temperature for activity was 45°C but the enzyme became unstable at temperatures above 40°C. FoPGM requires the presence of a divalent cation for its function with maximum activity being obtained with Co(2+). The apparent K(m) for Co(2+) was found to be 10 μM. The enzyme was also active with other divalent metal ions such as Mn(2+), Mg(2+), Ni(2+) and Ca(2+) but to a lesser extent. The following kinetic constants were determined: v(max), 0.74 μmol mg(protein)(-1)min(-1); k(cat), 44.2 min(-1); K(m)(G1P), 0.10mM; K(m)(G1,6 diP), 1.03 μM; k(cat)/K(m)(G1P), 443 mM(-1)min(-1) and k(cat)/K(m)(G1,6 diP), 42,860 mM(-1)min(-1). The enzyme was considered to follow a Ping Pong substituted enzyme or enzyme isomerization mechanism.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase

The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.     

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.     

Importance of product/reactant equilibration in the kinetics of the phosphoglucose isomerization reaction by differential stopped flow microcalorimetry

The kinetics for the isomerization of fructose-6-phosphate to glucose-6-phosphate (F6P --> G6P) by baker's yeast phosphoglucose isomerase (PGI) with regard to k(cat) and K(m) were determined from analysis of differential stopped flow microcalorimeter measurements using the integrated form of the Michaelis-Menten rate equation. Values for K(m) (F6P --> G6P) that were determined at pH 8.0 and ionic strength 0.1M at 293.4, 298.4, 303.4, and 311.5K exhibited a linear dependence on the substrate concentration at each temperature because of the substrate-product equilibrium. The minimum values for K(m) ranged from 2.62+/-0.55 mM at 293.4K to 7.8+/-4.8mM at 311.5K and were the same as the minimum values for the reverse reaction (G6P --> F6P) at 293.4 K and 298.4 K. Minimum values for k(cat) increased with temperature, from 2.78+/-0.34s(-1) at 293.4K to 11.4+/-1.0s(-1) at 311.5K, and for the reverse reaction, G6P --> F6P, from 0.852+/-0.086 s(-1) at 293.4K to 1.46+/-0.06s(-1) at 298.4K. The enzyme efficiency at 311.5K is close to the collision rate for a diffusion-controlled process in solution. The [F6P]/[G6P] equilibrium constants were determined from comparison of the values of k(cat) in both directions and were 0.307+/-0.053 at 293.4K and 0.395+/-0.033 at 298.4K. The heats of reaction in the F6P --> G6P direction increased from -8.96+/-0.26 kJmol(-1) at 311.5K to -8.27+/-0.40 kJmol(-1) at 293.4K, a value in fair agreement with 7.01+/-0.32 kJmol(-1) in the opposite G6P --> F6P direction.     

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).     

Type II isopentenyl diphosphate isomerase from Synechocystis sp. strain PCC 6803

Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His(6)-tagged protein was produced in Escherichia coli and purified by Ni(2+) chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg(2+) for activity; K(m)(IPP) was 52 microM, and k(cat)(IPP) was 0.23 s(-1).     

Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.     

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.     

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.     

Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.     

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.