Identification of tms-26 as an allele of the gcaD gene, which encodes N-acetylglucosamine 1-phosphate uridyltransferase in Bacillus subtilis.

The temperature-sensitive Bacillus subtilis tms-26 mutant strain was characterized biochemically and shown to be defective in N-acetylglucosamine 1-phosphate uridyltransferase activity. At the permissive temperature (34 degrees C), the mutant strain contained about 15% of the wild-type activity of this enzyme, whereas at the nonpermissive temperature (48 degrees C), the mutant enzyme was barely detectable. Furthermore, the N-acetylglucosamine 1-phosphate uridyltransferase activity of the tms-26 mutant strain was much more heat labile in vitro than that of the wild-type strain. The level of N-acetylglucosamine 1-phosphate, the substrate of the uridyltransferase activity, was elevated more than 40-fold in the mutant strain at the permissive temperature compared with the level in the wild-type strain. During a temperature shift, the level of UDP-N-acetylglucosamine, the product of the uridyltransferase activity, decreased much more in the mutant strain than in the wild-type strain. An Escherichia coli strain harboring the wild-type version of the tms-26 allele on a plasmid contained increased N-acetylglucosamine 1-phosphate uridyltransferase activity compared with that in the haploid strain. It is suggested that the gene for N-acetylglucosamine 1-phosphate uridyltransferase in B. subtilis be designated gcaD.     

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].     

Studies of propionate toxicity in Salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth

Salmonella enterica serovar Typhimurium LT2 showed increased sensitivity to propionate when the 2-methylcitric acid cycle was blocked. A derivative of a prpC mutant (which lacked 2-methylcitrate synthase activity) resistant to propionate was isolated, and the mutation responsible for the newly acquired resistance to propionate was mapped to the citrate synthase (gltA) gene. These results suggested that citrate synthase activity was the source of the increased sensitivity to propionate observed in the absence of the 2-methylcitric acid cycle. DNA sequencing of the wild-type and mutant gltA alleles revealed that the ATG start codon of the wild-type gene was converted to the rare GTG start codon in the revertant strain. This result suggested that lower levels of this enzyme were present in the mutant. Consistent with this change, cell-free extracts of the propionate-resistant strain contained 12-fold less citrate synthase activity. This was interpreted to mean that, in the wild-type strain, high levels of citrate synthase activity were the source of a toxic metabolite. In vitro experiments performed with homogeneous citrate synthase enzyme indicated that this enzyme was capable of synthesizing 2-methylcitrate from propionyl-CoA and oxaloacetate. This result lent further support to the in vivo data, which suggested that citrate synthase was the source of a toxic metabolite.     

Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h^–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low K_m values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher K_m values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V_max values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high K_m for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde. Received: 31 August 1995 / Accepted: 4 December 1995     

Xanthine dehydrogenase from Drosophila melanogaster: purification and properties of the wild-type enzyme and of a variant lacking iron-sulfur centers.

Xanthine dehydrogenase has been purified to homogeneity by conventional procedures from the wild-type strain of the fruit fly Drosophila melanogaster, as well as from a rosy mutant strain (E89----K, ry5231) known to carry a point mutation in the iron-sulfur domain of the enzyme. The wild-type enzyme had all the specific properties that are peculiar to the molybdenum-containing hydroxylases. It had normal contents of molybdenum, the pterin molybdenum cofactor, FAD, and iron-sulfur centers. EPR studies showed its molybdenum center to be quite indistinguishable from that of milk xanthine oxidase. As isolated, only about 10% of the enzyme was present in the functional form, with most or all of the remainder as the inactive desulfo form. It is suggested that this may be present in vivo. Extensive proteolysis accompanied by the development of oxidase activity took place during isolation, but dehydrogenase activity was retained. EPR properties of the reduced iron-sulfur centers, Fe-SI and Fe-SII, in the enzyme are very similar to those of the corresponding centers in milk xanthine oxidase. The E89----K mutant enzyme variant was in all respects closely similar to the wild-type enzyme, with the exception that it lacked both of the iron-sulfur centers. This was established both by its having the absorption spectrum of a simple flavoprotein and by the complete absence of EPR signals characteristic of iron-sulfur centers in the reduced enzyme. Despite the lack of iron-sulfur centers, the mutant enzyme had xanthine:NAD+ oxidoreductase activity indistinguishable from that of the wild-type enzyme. Stopped-flow measurements indicated that, as for the wild-type enzyme, reduction of the mutant enzyme was rate-limiting in turnover. Thus, the iron-sulfur centers appear irrelevant to the normal turnover of the wild-type enzyme with these substrates. However, activity to certain oxidizing substrates, particularly phenazine methosulfate, is abolished in the mutant enzyme variant. This is one of the first examples of deletion by genetic means of iron-sulfur centers from an iron-sulfur protein. The relevance of our findings both to the roles of iron-sulfur centers in other systems and to the nature of the oxidizing substrate for the Drosophila enzyme in vivo are briefly discussed.     

Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide to biotin and contains the molybdopterin guanine dinucleotide (MGD) cofactor as its sole prosthetic group. Comparison of the primary sequences of BSOR and the closely related enzyme dimethyl sulfoxide reductase (DMSOR) indicated a number of conserved residues, including an active-site tryptophan residue (W90), which has been suggested to be involved in hydrogen bonding to the oxo group on the Mo(VI) center in BSOR. Site-directed mutagenesis has been used to replace tryptophan 90 in BSOR with phenylalanine, tyrosine, and alanine residues to examine the role of this residue in catalysis. All three BSOR mutant proteins were purified to homogeneity and contained MGD. The mutant proteins retained very limited activity toward the oxidizing substrates tested, with W90F retaining the most activity (3.4% of wild type). All three W90 mutant proteins exhibited greatly reduced k(cat) values compared to that of the wild-type enzyme, which was accompanied by little change in K(mapp). In addition, the mutant proteins had perturbed visible absorption and circular dichroism spectra suggesting different oxidation states of the Mo center. Purified samples of wild-type BSOR did not exhibit electron paramagnetic resonance (EPR) signals indicating a Mo(VI) center. After redox-cycling, partially reduced samples of wild-type BSOR revealed a proton-split S=1/2 Mo(V) resonance (g(1,2,3)=1.999, 1.981, 1.967; A(1,2,3)=1.40, 1.00, 1.05 mT) analogous to that observed in DMSOR. In contrast, EPR studies of the purified W90 mutant proteins revealed distinct S=1/2 Mo(V) resonances that were resistant to both oxidation and reduction, indicating that the Mo was trapped in the intermediate Mo(V) oxidation state. These results strongly suggest that W90 in BSOR plays a critical role in catalysis by serving as a hydrogen bond donor to the oxo group on the Mo(VI) center.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-beta-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more beta-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in beta-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.     

Heterologous expression and characterization of wild-type human cytochrome P450 1A2 without conventional N-terminal modification in Escherichia coli

In this study, wild-type human CYP1A2 without the conventional N-terminal modification (second codon GCT) or the truncation of the N-terminal hydrophobic region was functionally expressed in Escherichia coli. Its enzymatic properties were compared with N-terminally modified CYP1A2. Although modified CYP1A2 is almost all high-spin, some wild-type CYP1A2 shifted to low-spin. Spectral binding titrations with several ligands could be performed with wild-type enzyme, but not with modified enzyme. Kinetic parameters for several substrates were similar for the two CYP1A2 enzymes. However, the oxidation rates of phenacetin by modified enzyme were approximately 2-fold higher than those by wild-type enzyme. The intermolecular isotope effects were approximately 2 for phenacetin O-deethylation catalyzed by both enzymes. However, the wild-type enzyme, but not the modified enzyme, increased C-hydroxylation when O-deethylation rates were lowered by deuterium substitution. Molecular switching indicates that phenacetin rotates within the active site of wild-type enzyme and suggests a looser conformation in the active site of the wild-type enzyme than of the modified enzyme. These results reveal that the overall enzymatic properties of wild-type CYP1A2 enzyme are quite similar to those of modified CYP1A2, although its active site environment seems to differ from that of the modified enzyme.     

The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenes DeltanrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.     

Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases

The kinetics of flavin reduction in two mutant forms of human cytochrome P450 reductase have been studied by stopped-flow spectroscopy with absorption and fluorescence detection. The mutant enzymes were altered at the position of Trp-676, which, by analogy with the structure of rat CPR, is close to the isoalloxazine ring of the enzyme-bound FAD. We show that mutant CPRs in which Trp-676 has been changed to histidine (W676H) and alanine (W676A) can be reduced by NADPH only to the two-electron level in single mixing stopped-flow experiments. The concentration dependence of the rate of hydride transfer indicates that the second, noncatalytic NADPH-binding site present in wild-type CPR is retained in the mutant enzymes. Detailed studies of W676H CPR indicate that further reduction of the enzyme beyond the two electron level is prevented due to the slow release of NADP(+) from the active site following the first hydride transfer from NADPH, owing to the stability of a reduced enzyme-NADP(+) charge-transfer complex. Reduction to the four-electron level is achieved in a sequential mixing stopped-flow experiment. In this procedure, W676H CPR is reacted first with a stoichiometric amount of NADPH, and then, following a delay of 100 ms, with excess NADPH. The data indicate that occupancy of the noncatalytic coenzyme site also hinders NADP(+) release from reduced enzyme. Fluorescence stopped-flow studies of the W676H and wild-type CPR enzymes reveal that the complex signals associated with reduction of wild-type CPR by NADPH are attributable to changes in the environment of residue W676. From these studies, a model is proposed for nicotinamide binding in wild-type CPR. In this model W676 serves as a trigger to release NADP(+) from the active site following hydride transfer. In the W676H enzyme, the slow release of NADP(+) is a consequence of the combined effects of (i) removing W676 by mutagenesis (thus removing the trigger for displacement) and (ii) the binding of NADPH in the noncatalytic site, thus trapping NADP(+) in the catalytic site.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-β-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more β-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in β-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Phosphorescence and optically detected magnetic resonance measurements of the 2'AMP and 2'GMP complexes of a mutant ribonuclease T1 (Y45W) in solution: correlation with X-ray crystal structures.

Phosphorescence and ODMR measurements have been made on ribonuclease T1 (RNase T1), the mutated enzyme RNase T1 (Y45W), and their complexes with 2'GMP and 2'AMP. It is not possible to observe the phosphorescence of Trp45 in RNase T1 (Y45W). Only that of the naturally occurring Trp59 is seen. The binding of 2'GMP to wild-type RNase T1 produces only a minor red shift in the phosphorescence and no change in the ODMR spectrum of Trp59. However, a new tryptophan 0,0-band is found 8.2 nm to the red of the Trp59 0,0-band in the 2'GMP complex of the mutated RNase T1 (Y45W). Wavelength-selected ODMR measurements reveal that the red-shifted emission induced by 2'GMP binding, assigned to Trp45, occurs from a residue with significantly different zero-field splittings than those of Trp59, a buried residue subject to local polar interactions. The phosphorescence red shift and the zero-field splitting parameters demonstrate that Trp45 is located in a polarizable environment in the 2'GMP complex. In contrast with 2'GMP, binding of 2'AMP to RNase T1 (Y45W) induces no observable phosphorescence emission from Trp45, but leads only to a minor red shift in the phosphorescence origin of Trp59 in both the mutated and wild-type enzyme. The lack of resolved phosphorescence emission from Trp45 in RNase T1 (Y45W) implies that the emission of this residue is quenched in the uncomplexed enzyme. We conclude that local conformational changes that occur upon binding 2'GMP remove quenching residues from the vicinity of Trp45, restoring its luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity.

The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.     

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.     

Characterization and high-level production of D-amino acid oxidase in Candida boidinii

D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host. The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor. The enzyme was active toward several D-amino acids such as D-Ala, D-Met, and D-Ser. An alcohol oxidase-depleted strain (aod1delta) was found to be a more suitable host for DAO production than the wild-type strain. Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1delta strain. Finally, an aod1delta strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30% of the total soluble proteins.