Proteome changes after metabolic engineering to enhance aerobic mineralization of cis-1,2-dichloroethylene

Metabolically engineered Escherichia coli has previously been used to degrade cis-1,2-dichloroethylene (cis-DCE). The strains express the six genes of an evolved toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green, which formed a reactive epoxide) with either (1) gamma-glutamylcysteine synthetase (GSHI, which forms glutathione) and the glutathione S-transferase IsoILR1 from Rhodococcus AD45 (which adds glutathione to the reactive cis-DCE epoxide) or (2) with an evolved epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA F108L/I219L/C248I which converts the reactive cis-DCE epoxide to a diol). Here, the impact of this metabolic engineering for bioremediation was assessed by investigating the changes in the proteome through a quantitative shotgun proteomics technique (iTRAQ) by tracking the changes due to the sequential addition of TOM-Green, IsoILR1, and GSHI and due to adding the evolved EchA versus the wild-type enzyme to TOM-Green. For the TOM-Green/EchA system, 8 proteins out of 268 identified proteins were differentially expressed in the strain expressing EchA F108L/I219L/C248I relative to wild-type EchA (e.g., EchA, protein chain elongation factor EF-Ts, 50S ribosomal subunits L7/L12/L32/L29, cysteine synthase A, glycerophosphodiester phosphodiesterase, iron superoxide dismutase). For the TOM-Green/IsoILR1/GSHI system, the expression level of 49 proteins was changed out of 364 identified proteins. The induced proteins due to the addition of TOM-Green, IsoILR1, and GSHI were involved in the oxidative defense mechanism, pyruvate metabolism, and glutathione synthesis (e.g., 30S ribosomal subunit proteins S3 and S16, 50S ribosomal subunit protein L20, alkyl hydroperoxide reductase, lactate dehydrogenase, acetate kinase, cysteine synthase A). Enzymes involved in indole synthesis, fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle were repressed (e.g., tryptophanase, acetyl-CoA carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase). Hence, the metabolic engineering that leads to enhanced aerobic degradation of 1 mM cis-DCE (2.4-4-fold more chloride ions released) and reduced toxicity from cis-DCE epoxide results in enhanced synthesis of glutathione coupled with an induced stress response as well as repression of fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle.     

Protein engineering of epoxide hydrolase from Agrobacterium radiobacter AD1 for enhanced activity and enantioselective production of (R)-1-phenylethane-1,2-diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 +/- 0.09 versus 1.04 +/- 0.07 micromol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 +/- 2 versus 2.4 +/- 0.3 micromol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 +/- 0.5 versus 2.6 +/- 0.0 micromol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 +/- 0.3 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 +/- 0.8 versus 1.8 +/- 0.2 micromol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.     

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Metabolic pathway engineering to enhance aerobic degradation of chlorinated ethenes and to reduce their toxicity by cloning a novel glutathione S-transferase, an evolved toluene o-monooxygenase, and gamma-glutamylcysteine synthetase

Aerobic, co-metabolic bioremediation of trichloroethylene (TCE), cis-1,2-dichloroethylene (cis-DCE) and other chlorinated ethenes with monooxygenase-expressing microorganisms is limited by the toxic epoxides produced as intermediates. A recombinant Escherichia coli strain less sensitive to the toxic effects of cis-DCE, TCE and trans-1,2-dichloroethylene (trans-DCE) degradation has been created by engineering a novel pathway consisting of eight genes including a DNA-shuffled toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green), a newly discovered glutathione S-transferase (GST) from RhodococcusAD45 (IsoILR1), found to have activity towards epoxypropane and cis-DCE epoxide, and an overexpressed E. coli mutant gamma-glutamylcysteine synthetase (GSHI*). Along with IsoILR1, another new RhodococcusAD45 GST, IsoILR2, was cloned that lacks activity towards cis-DCE epoxide and differs from IsoILR1 by nine amino acids. The recombinant strain in which TOM-Green and IsoILR1 were co-expressed on separate plasmids degraded 1.9-fold more cis-DCE compared with a strain that lacked IsoILR1. In the presence of IsoILR1 and TOM-Green, the addition of GSH1* resulted in a sevenfold increase in the intracellular GSH concentration and a 3.5-fold improvement in the cis-DCE degradation rate based on chloride released (2.1 +/- 0.1 versus 0.6 +/- 0.1 nmol min(-1) mg(-1) protein at 540 microM), a 1.8-fold improvement in the trans-DCE degradation rate (1.29 +/- 0.03 versus 0.71 +/- 0.04 nmol x min(-1) mg(-1) protein at 345 microM) and a 1.7-fold improvement in the TCE degradation rate (6.8 +/- 0.24 versus 4.1 +/- 0.16 nmol x min(-1) mg(-1) protein at 339 microM). For cis-DCE degradation with TOM-Green (based on substrate depletion), V(max) was 27 nmol x min(-1) mg(-1) protein with both IsoILR1 and GSHI* expressed compared with V(max) = 10 nmol x min(-1) mg(-1) protein for the GST(-)GSHI*(-) strain. In addition, cells expressing IsoILR1 and GSHI* grew 78% faster in rich medium than a strain lacking these two heterologous genes.     

Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol.

To study membrane topology and mechanism for substrate specificity, we truncated residues 2-24 in microsomal cytochrome P450 7A1 (P450 7A1) and introduced conservative and nonconservative substitutions at positions 214-227. Heterologous expression in Escherichia coli was followed by investigation of the subcellular distribution of the mutant P450s and determination of the kinetic and substrate binding parameters for cholesterol. The results indicate that a hydrophobic region, comprising residues 214-227, forms a secondary site of attachment to the membrane in P450 7A1 in addition to the NH(2)-terminal signal-anchor sequence. There are two groups of residues at this enzyme-membrane interface. The first are those whose mutation results in more cytosolic P450 (Val-214, His-225, and Met-226). The second group are those whose mutation leads to more membrane-bound P450 (Phe-215, Leu-218, Ile-224, and Phe-227). In addition, the V214A, V214L, V214T, F215A, F215L, F215Y, L218I, L218V, V219T, and M226A mutants showed a 5-12-fold increased K(m) for cholesterol. The k(cat) of the V214A, V214L, V219T, and M226A mutants was increased up to 1.8-fold, and that of the V214T, F215A, F215L, F215Y, L218I, and L218V mutants was decreased 3-10.5-fold. Based on analysis of these mutations we suggest that cholesterol enters P450 7A1 through the membrane, and Val-214, Phe-215, and Leu-218 are the residues located near the point of cholesterol entry. The results provide an understanding of both the P450 7A1-membrane interactions and the mechanism for substrate specificity.     

Molecular epidemiology of drug resistance markers of Plasmodium falciparum in Yunnan Province, China

ABSTRACT: BACKGROUND: The mutations in Plasmodium falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr), dihydropteroate synthase (pfdhps) and ATPase (pfatp6) genes were associated with anti-malaria drug resistance. The aim of this study was to investigate the prevalence of polymorphisms in pfcrt, pfmdr1, pfdhfr, pfdhps and pfatp6 in Yunnan Province. Finger-prick blood samples were collected from malaria-positive patients from Yunnan Province in 2009-2010. Single-nucleotide polymorphisms (SNPs) in the resistance-related genes were analysed by various PCR-based methods. RESULTS: A total of 108 blood samples were collected. Although chloroquine has not been used to treat falciparum malaria for nearly 30 years, 95.3% of the parasites still carried the pfcrt K76T mutation, whereas the majority of isolates displayed the wild-type pfmdr1 N86 and D1246 sequences. The molecular level of sulphadoxine-pyrimethamine resistance in P. falciparum was high. The most prevalent mutation was pfdhfr C59R (95.9%), whereas the frequencies of the quadruple, triple and double mutants were 22.7% (N51I/C59R/S108N/I164L), 51.5% (N51I/C59R/S108N, N51I/C59R/I164L and C59R/S108N/ I164L) and 21.6% (N51I/ C59R, C59R/S108N and C59R/I164L), respectively. A437G (n=77) and K540E (n=71) were the most prevalent mutations in pfdhps, and 52.7% of the samples were double mutants, among which A437G/K540E was the most common double mutation (37/49). Quadruple mutants were found in 28.0% (26/93) of samples. A total of 8.6% of isolates (8/93) carried the S436A/A437G/A581G triple mutation. No mutations were found in pfatp6 codons 623 or 769, but another two mutations (N683K and R756K) were found in 4.6% (3/97) and 9.2% (6/97) of parasite isolates, respectively. CONCLUSIONS: This study identified a high frequency of mutations in pfcrt, pfdhfr and pfdhps associated with CQ and SP resistance in P. falciparum and no mutations linked to artemisinin resistance (pfatp6). Molecular epidemiology should be included in routine surveillance protocols and used to provide complementary information to assess the appropriateness of the current national anti-malarial drug policy.     

Increasing hydrophobicity or disulfide bridging at the factor VIII A1 and C2 domain interface enhances procofactor stability

Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ～3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ～4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ～4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.     

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining the Solanum tuberosum and an evolved Agrobacterium radiobacter AD1 epoxide hydrolases

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.     

Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations

Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.     

SCH28080, a K+-competitive inhibitor of the gastric H,K-ATPase,binds near the M5-6 luminal loop,preventing K+ access to the ion binding domain

Inhibition of the gastric H,K-ATPase by the imidazo[1,2-alpha]pyridine, SCH28080, is strictly competitive with respect to K+ or its surrogate, NH4+. The inhibitory kinetics [V(max), K(m,app)(NH4+), K(i)(SCH28080), and competitive, mixed, or noncompetitive] of mutants can define the inhibitor binding domain and the route to the ion binding region within M4-6. While mutations Y799F, Y802F, I803L, S806N, V807I (M5), L811V (M5-6), Y928H (M8), and Q905N (M7-8) had no effect on inhibitor kinetics, mutations P798C, Y802L, P810A, P810G, C813A or -S, I814V or -F, F818C, T823V (M5, M5-6, and M6), E914Q, F917Y, G918E, T929L, and F932L (M7-8 and M8) reduced the affinity for SCH28080 up to 10-fold without affecting the nature of the kinetics. In contrast, the L809F substitution in the loop between M5 and M6 resulted in an approximately 100-fold decrease in inhibitor affinity, and substitutions L809V, I816L, Y925F, and M937V (M5-6, M6, and M8) reduced the inhibitor affinity by 10-fold, all resulting in noncompetitive kinetics. The mutants L811F, Y922I, and I940A also reduced the inhibitor affinity up to 10-fold but resulted in mixed inhibition. The mutations I819L, Q923V, and Y925A also gave mixed inhibition but without a change in inhibitor affinity. These data, and the 9-fold loss of SCH28080 affinity in the C813T mutant, suggest that the binding domain for SCH28080 contains the surface between L809 in the M5-6 loop and C813 at the luminal end of M6, approximately two helical turns down from the ion binding region, where it blocks the normal ion access pathway. On the basis of a model of the Ca-ATPase in the E2 conformation (PDB entry 1kju), the mutants that change the nature of the kinetics are arranged on one side of M8 and on the adjacent side of the M5-6 loop and M6 itself. This suggests that mutations in this region modify the enzyme structure so that K+ can access the ion binding domain even with SCH28080 bound.     

Protein engineering of hydrogenase 3 to enhance hydrogen production

The large subunit (HycE, 569 amino acids) of Escherichia coli hydrogenase 3 produces hydrogen from formate via its Ni–Fe-binding site. In this paper, we engineered HycE for enhanced hydrogen production by an error-prone polymerase chain reaction (epPCR) using a host that lacked hydrogenase activity via the hyaB hybC hycE mutations. Seven enhanced HycE variants were obtained with a novel chemochromic membrane screen that directly detected hydrogen from individual colonies. The best epPCR variant contained eight mutations (S2T, Y50F, I171T, A291V, T366S, V433L, M444I, and L523Q) and had 17-fold higher hydrogen-producing activity than wild-type HycE. In addition, this variant had eightfold higher hydrogen yield from formate compared to wild-type HycE. Deoxyribonucleic acid shuffling using the three most-active HycE variants created a variant that has 23-fold higher hydrogen production and ninefold higher yield on formate due to a 74-amino acid carboxy-terminal truncation. Saturation mutagenesis at T366 of HycE also led to increased hydrogen production via a truncation at this position; hence, 204 amino acids at the carboxy terminus may be deleted to increase hydrogen production by 30-fold. This is the first random protein engineering of a hydrogenase.     

A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.     

A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach

The redox potential of cytochromes sets the energy yield possible in metabolism and is also a key determinant of the rate at which redox reactions proceed. Here, the heme protein, cytochrome b(562), is used to study the in vitro evolution of redox potential within a library of variants containing the same structural archetype, the four-helix bundle. Multisite variations in the active site of cytochrome b(562) were introduced. A library of variants containing random mutations in place of R98 and R106 was created, and the redox potentials of a statistical sampling of this library were measured. This procedure was carried out for both the low- and high-potential variants of a previously studied F61X/F65X, first-generation library [Springs, S. L., Bass, S. E., and McLendon, G. L. (2000) Biochemistry 39, 6075]. The second-generation library reported here has a range of redox potentials which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands (but different folds) and exceeds the range exhibited phylogenetically by the cytochrome c' family which internally maintains the same axial ligation and fold. A statistical analysis of the libraries examined reveals that the redox potential of WT cyt b(562) is found at the high-potential extremum of the distribution, indicating that this protein apparently evolved to differentially stabilize the reduced protein. The 2.7 A crystal structure of F61I/F65Y/R106L (low-potential variant of the second-generation library) was solved and is compared to the wild-type structure and the 2.2 A resolution structure of the F61I/F65Y variant (low-potential variant of the first-generation library). The structures indicate that charge-dipole effects are responsible for shifting the redox equilibrium toward the oxidized state in both the F61I/F65Y and F61I/F65Y/R106L variants. Specifically, a new protein dipole is introduced into the heme microenvironment as a result of the F65Y mutation, two new internal water molecules (one in hydrogen-bonding distance of Y65) are found, and in the case of F61I/F65Y/R106L (DeltaE(m) = 158 mV vs NHE), increased solvent exposure of the heme as a result of the R106L substitution is identified.     

Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the α/β-fold epoxide hydrolase Alp1U

Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.     

Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1

A recombinant strain of Escherichia coli (JM109/pBZ1260) expressing constitutively toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 degraded binary mixtures (100 microM each) of tetrachloroethylene (PCE) with either trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), or vinyl chloride (VC). PCE degradation was 8-20% for these binary mixtures, while TCE and trans-DCE with PCE were degraded at 19%, 1,1-DCE at 37%, cis-DCE at 97%, and VC at 27%. The host P. stutzeri OXI was also found to degrade binary mixtures of PCE/TCE, PCE/cis-DCE, and PCE/VC when induced with toluene. Degradation of quaternary mixtures of PCE/TCE/trans-DCE/VC and PCE/TCE/cis-DCE/VC by JM109/pBZ1260 were also investigated as well as mixtures of PCE/TCE/trans-DCE/1,1-DCE/cis-DCE/VC; when all the chlorinated compounds were present, the best degradation occurred with 24-51% removal of each. For these degradation reactions, 39-85% of the stoichiometric chloride expected from complete degradation of the chlorinated ethenes was detected. The time course of PCE/TCE/1,1-DCE degradation was also measured for a mixture of 8, 17, and 6 microM, respectively; initial degradation rates were 0.015, 0.023. and 0.029 nmol/min x mg protein, respectively. This indicates that for the first time an aerobic enzyme can degrade mixtures of all chlorinated ethenes, including the once--so it was believed-completely recalcitrant PCE.     

Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active- and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6(54/84) variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystal-structure analysis of the V6(54/84) variant bound to ritonavir and indinavir shows structural changes in the 80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggest possible changes in the dynamics of flap opening and closing.     

Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method

α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.