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1.
Biochemistry ; 56(50): 6521-6532, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29087185

RESUMO

A review of the previous stabilization of α/ß-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model α/ß-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). All strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus. A web-based automated program that predicts substitutions needed to mutate to consensus is available at http://kazlab.umn.edu .


Assuntos
Hidrolases/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Estabilidade Enzimática/fisiologia , Modelos Moleculares , Mutagênese , Mutação , Mutação Puntual , Proteínas/genética , Proteínas/metabolismo
2.
Mol Biol Evol ; 33(4): 971-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26681154

RESUMO

The means by which superfamilies of specialized enzymes arise by gene duplication and functional divergence are poorly understood. The escape from adaptive conflict hypothesis, which posits multiple copies of a gene encoding a primitive inefficient and highly promiscuous generalist ancestor, receives support from experiments showing that resurrected ancestral enzymes are indeed more substrate-promiscuous than their modern descendants. Here, we provide evidence in support of an alternative model, the innovation-amplification-divergence hypothesis, which posits a single-copied ancestor as efficient and specific as any modern enzyme. We argue that the catalytic mechanisms of plant esterases and descendent acetone cyanohydrin lyases are incompatible with each other (e.g., the reactive substrate carbonyl must bind in opposite orientations in the active site). We then show that resurrected ancestral plant esterases are as catalytically specific as modern esterases, that the ancestor of modern acetone cyanohydrin lyases was itself only very weakly promiscuous, and that improvements in lyase activity came at the expense of esterase activity. These observations support the innovation-amplification-divergence hypothesis, in which an ancestor gains a weak promiscuous activity that is improved by selection at the expense of the ancestral activity, and not the escape from adaptive conflict in which an inefficient generalist ancestral enzyme steadily loses promiscuity throughout the transition to a highly active specialized modern enzyme.


Assuntos
Evolução Molecular , Variação Genética , Hidrolases/genética , Filogenia , Aldeído Liases/genética , Catálise , Domínio Catalítico , Duplicação Gênica
3.
J Am Chem Soc ; 138(3): 1046-56, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26736133

RESUMO

Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/ß-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.


Assuntos
Aldeído Liases/metabolismo , Biocatálise , Esterases/metabolismo , Aldeído Liases/química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Esterases/química , Ésteres/química , Ésteres/metabolismo , Cianeto de Hidrogênio/química , Cianeto de Hidrogênio/metabolismo , Hidrólise , Nitrilas/química , Nitrilas/metabolismo
4.
Biotechnol Bioeng ; 113(7): 1493-503, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26693833

RESUMO

p-Coumaric acid (pCA) is abundant in biomass with low lignin content, such as straw and stubble from rye, wheat, and barley. pCA can be isolated from biomass and used for the synthesis of aromatic hydrocarbons. Here, we report engineering of the natural pathway for conversion of pCA into p-hydroxybenzoic acid (pHBA) to increase the amount of pHBA that accumulates more than 100-fold. Burkholderia glumae strain BGR1 (BGR1) grows efficiently on pCA as a sole carbon source via a CoA-dependent non-ß-oxidation pathway. This pathway removes two carbons from pCA as acetyl-CoA yielding p-hydroxybenzaldehyde and subsequently oxidizes it to pHBA. To increase the amount of accumulated pHBA in BGR1, we first deleted two genes encoding enzymes that degrade pHBA in the ß-ketoadipate pathway. At 10 mM of pCA, the double deletion mutant BGR1_PB4 (Δphb3hΔbcl) accumulated pHBA with 95% conversion, while the control BGR1 accumulated only with 11.2% conversion. When a packed bed reactor containing immobilized BGR1_PB4 cells was operated at a dilution rate 0.2 h(-1) , the productivity of pHBA was achieved at 9.27 mg/L/h for 134 h. However, in a batch reactor at 20 mM pCA, growth of BGR1_PB4 was strongly inhibited, resulting in a low conversion of 19.3%. To further increase the amount of accumulated pCA, we identified the first enzyme in the pathway, p-hydroxcinnmaoyl-CoA synthetase II (phcs II), as the rate-limiting enzyme. Over expression of phcs II using a Palk promoter in a batch reaction at 20 mM of pCA yielded 99.0% conversion to pHBA, which is the highest concentration of pHBA ever reported using a biological process. Biotechnol. Bioeng. 2016;113: 1493-1503. © 2015 Wiley Periodicals, Inc.


Assuntos
Burkholderia/metabolismo , Ácidos Cumáricos/metabolismo , Engenharia Metabólica/métodos , Parabenos/metabolismo , Burkholderia/genética , Ácidos Cumáricos/análise , Lignina , Mutação , Parabenos/análise , Propionatos
5.
Biochemistry ; 54(28): 4330-41, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26110207

RESUMO

α/ß-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. We investigated a plant esterase, salicylic acid binding protein 2 (SABP2), as a model α/ß-hydrolase. SABP2 shows typical stability to urea (unfolding free energy 6.9 ± 1.5 kcal/mol) and to heat inactivation (T1/2 15min 49.2 ± 0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homologue or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1), and E215P (+0.9). Introducing proline in the cap domain did not stabilize SABP2 (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/2 15min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P−S70P ΔT1/2 15min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/ß-hydrolases.


Assuntos
Esterases/química , Hidrolases/química , Nicotiana/química , Proteínas de Plantas/química , Prolina/química , Substituição de Aminoácidos , Domínio Catalítico , Estabilidade Enzimática , Esterases/genética , Hidrolases/genética , Modelos Moleculares , Proteínas de Plantas/genética , Prolina/genética , Conformação Proteica , Desnaturação Proteica , Desdobramento de Proteína , Nicotiana/genética
6.
Chembiochem ; 15(13): 1931-8, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25044660

RESUMO

The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a kcat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.


Assuntos
Acetonitrilas/química , Aldeído Liases/química , Esterases/química , Hevea/enzimologia , Hidrocarbonetos Aromáticos/química , Aldeído Liases/genética , Catálise , Domínio Catalítico/genética , Ésteres/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Desdobramento de Proteína , Estereoisomerismo , Especificidade por Substrato
7.
Chembiochem ; 14(10): 1231-8, 2013 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-23780920

RESUMO

A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to COH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examined for O-dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A-ring inactivated in order to find enhanced 3'-regioselectiviy). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L(-1) production titer and greatly increased 3'-regioselectiviy (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.


Assuntos
Colorimetria/métodos , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Catálise , Sistema Enzimático do Citocromo P-450/química , Remoção de Radical Alquila , Hidroxilação , Oxirredução , Especificidade por Substrato
8.
Chemistry ; 19(9): 3037-46, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23325572

RESUMO

Some serine hydrolases also catalyze a promiscuous reaction--reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five X-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k(cat) comparison) than the wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over the wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83-times faster (k(cat) comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE (ß(0)=170 vs. 160 M(-1)), and a similar fast formation of acetyl-enzyme (140 vs. 62 U mg(-1)). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II ß-turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I ß-turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE, so that an acetate accepts a hydrogen bond to promote faster formation of the acetyl-enzyme.


Assuntos
Ácidos Carboxílicos/química , Hidrolases de Éster Carboxílico/química , Esterases/química , Prolina/química , Pseudomonas fluorescens/enzimologia , Sítios de Ligação , Hidrolases de Éster Carboxílico/metabolismo , Catálise , Cristalografia por Raios X , Esterases/metabolismo , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Estrutura Molecular , Engenharia de Proteínas , Água/química
9.
Appl Microbiol Biotechnol ; 97(4): 1501-11, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23081771

RESUMO

Phenolic acid decarboxylase (PAD) catalyzes the non-oxidative decarboxylation of p-coumaric acid (pCA) to p-hydroxystyrene (pHS). PAD from Bacillus amyloliquefaciens (BAPAD), which showed k (cat)/K (m) value for pCA (9.3 × 10³ mM⁻¹ s⁻¹), was found as the most active one using the "Subgrouping Automata" program and by comparing enzyme activity. However, the production of pHS of recombinant Escherichia coli harboring BAPAD showed only a 22.7 % conversion yield due to product inhibition. Based on the partition coefficient of pHS and biocompatibility of the cell, 1-octanol was selected for the biphasic reaction. The conversion yield increased up to 98.0 % and 0.83 g/h/g DCW productivity was achieved at 100 mM pCA using equal volume of 1-octanol as an organic solvent. In the optimized biphasic reactor, using a three volume ratio of 1-octanol to phosphate buffer phase (50 mM, pH 7.0), the recombinant E. coli produced pHS with a 88.7 % conversion yield and 1.34 g/h/g DCW productivity at 300 mM pCA.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Carboxiliases/metabolismo , Ácidos Cumáricos/metabolismo , Estirenos/metabolismo , Bacillus/química , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Biotransformação , Carboxiliases/química , Carboxiliases/genética , Ácidos Cumáricos/química , Cinética , Estirenos/química
10.
Appl Environ Microbiol ; 78(3): 684-94, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22101053

RESUMO

7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and ß subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 µM) 7-OMA upon supplementation with 500 µM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 µM) 7-OMA from 500 µM NRN in 24 h.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Flavonoides/metabolismo , Engenharia Metabólica , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Ácidos Cumáricos/metabolismo , Medicago sativa/enzimologia , Medicago sativa/genética , Nocardia/enzimologia , Nocardia/genética , Petroselinum/enzimologia , Petroselinum/genética , Petunia/enzimologia , Petunia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Propionatos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética
11.
Chemistry ; 18(26): 8130-9, 2012 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-22618813

RESUMO

Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (ß(0)=170 M(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (ß(0)=330 M(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by (18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T(d)1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.


Assuntos
Ácidos Carboxílicos/química , Peróxido de Hidrogênio/química , Modelos Químicos , Pseudomonas fluorescens/enzimologia , Serina Proteases/metabolismo , Acetatos/metabolismo , Catálise , Simulação por Computador , Cinética , Estrutura Molecular , Serina Proteases/química , Água/química
12.
Int J Biol Macromol ; 222(Pt B): 2341-2352, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36216104

RESUMO

Cyclization of proteins using SpyTag/SpyCatcher is a novel approach to increase their thermal stability. In this paper, we test this approach on two ß-galactosidases from Bacillus circulans, BgaB and BgaC, and find that BgaB was stabilized while BgaC was not. Wild-type BgaB precipitated completely upon heating above 70 °C, but after SpyRing cyclization, it remained soluble after heating to 90 °C. Similarly, wild-type BgaB retained only 50 % activity after heating at 60 °C for 10 min, but this increased to 80 % after SpyRing cyclization. In contrast, cyclization decreased the stability of BgaC. After SpyRing cyclization, BgaC only retained 2 % activity after 20-min incubation at 55 °C, whereas the wild-type BgaC retained 25 % activity. One reason for the different effect of cyclization may the shorter distance between the N- and C-termini in BgaB (20.2 Å) as compared to BgaC (43.7 Å). The intrinsic fluorescence and circular dichroism spectra suggested that SpyRing cyclization of BgaB did not significantly change its conformation or secondary structure. SpyRing cyclized BgaB yielded similar amounts and compositions of galacto-oligosaccharides using a high initial lactose concentration (40 %, w/v), but a slightly higher amount at low initial lactose concentration (5 %, w/v) suggesting increased transgalactosylation activity.


Assuntos
Lactose , Oligossacarídeos , Ciclização , Lactose/metabolismo , beta-Galactosidase/química , Galactose
13.
Chembiochem ; 12(5): 768-76, 2011 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-21351219

RESUMO

Acyl transfer is a key reaction in biosynthesis, including synthesis of antibiotics and polyesters. Although researchers have long recognized the similar protein fold and catalytic machinery in acyltransferases and hydrolases, the molecular basis for the different reactivity has been a long-standing mystery. By comparison of X-ray structures, we identified a different oxyanion-loop orientation in the active site. In esterases/lipases a carbonyl oxygen points toward the active site, whereas in acyltransferases a NH of the main-chain amide points toward the active site. Amino acid sequence comparisons alone cannot identify such a difference in the main-chain orientation. To identify how this difference might change the reaction mechanism, we solved the X-ray crystal structure of Pseudomonas fluorescens esterase containing a sulfonate transition-state analogue bound to the active-site serine. This structure mimics the transition state for the attack of water on the acyl-enzyme and shows a bridging water molecule between the carbonyl oxygen mentioned above and the sulfonyl oxygen that mimics the attacking water. A possible mechanistic role for this bridging water molecule is to position and activate the attacking water molecule in hydrolases, but to deactivate the attacking water molecule in acyl transferases.


Assuntos
Aciltransferases/química , Haemophilus influenzae/enzimologia , Hidrolases/química , Pseudomonas fluorescens/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Esterases/química , Modelos Moleculares , Conformação Proteica
14.
Chembiochem ; 12(10): 1508-17, 2011 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-21506229

RESUMO

The superfamily of α/ß-hydrolase fold enzymes is one of the largest known protein families, including a broad range of synthetically useful enzymes such as lipases, esterases, amidases, hydroxynitrile lyases, epoxide hydrolases and dehalogenases. This minireview covers methods developed for efficient protein engineering of these enzymes. Special emphasis is placed on the alteration of enzyme properties such as substrate range, thermostability and enantioselectivity for their application in biocatalysis. In addition, concepts for the investigation of the evolutionary relationship between the different members of this protein superfamily are covered, together with successful examples.


Assuntos
Bactérias/enzimologia , Evolução Molecular Direcionada/métodos , Fungos/enzimologia , Hidrolases/química , Hidrolases/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Bactérias/química , Bactérias/genética , Fungos/química , Fungos/genética , Hidrolases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência
15.
Sci Adv ; 7(29)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34272238

RESUMO

Extensive exploration of a protein's sequence space for improved or new molecular functions requires in vivo evolution with large populations. But disentangling the evolution of a target protein from the rest of the proteome is challenging. Here, we designed a protein complex of a targeted artificial DNA replisome (TADR) that operates in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of the target plasmid up to 2.3 × 105-fold with only a 78-fold increase in off-target mutagenesis. It was used to evolve itself to increase error rate and increase the efficiency of an efflux pump while simultaneously expanding the substrate repertoire. TADR enables multiple simultaneous substitutions to discover functions inaccessible by accumulating single substitutions, affording potential for solving hard problems in molecular evolution and developing biologic drugs and industrial catalysts.


Assuntos
DNA Polimerase Dirigida por DNA , Complexos Multienzimáticos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutagênese , Plasmídeos/genética
16.
Biochemistry ; 49(9): 1931-42, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20112920

RESUMO

Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.


Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Peróxido de Hidrogênio/química , Pseudomonas fluorescens/enzimologia , Ácido Acético/química , Substituição de Aminoácidos/genética , Proteínas de Bactérias/genética , Biocatálise , Caproatos/química , Hidrolases de Éster Carboxílico/genética , Domínio Catalítico/genética , Cristalização , Cristalografia por Raios X , Difusão , Ésteres/química , Hidrólise , Lactonas/química , Leucina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Prolina/genética , Pseudomonas fluorescens/genética
17.
Methods Enzymol ; 643: 129-148, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32896278

RESUMO

The consensus sequence approach to predicting stabilizing substitutions in proteins rests on the notion that conserved amino acids are more likely to contribute to the stability of a protein fold than non-conserved amino acids. To implement a prediction for a target protein sequence, one finds homologous sequences and aligns them in a multiple sequence alignment. The sequence of the most frequently occurring amino acid at each position is the consensus sequence. Replacement of a rarely occurring amino acid in the target with a frequently occurring amino acid from the consensus sequence is predicted to be stabilizing. Consensus Finder is an open-source web tool that automates this prediction. This chapter reviews the rationale for the consensus sequence approach and explains the options for fine-tuning this approach using Staphylococcus nuclease A as an example.


Assuntos
Proteínas , Sequência de Aminoácidos , Consenso , Sequência Consenso , Proteínas/genética , Alinhamento de Sequência
18.
PLoS One ; 15(6): e0235341, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603354

RESUMO

Hydroxynitrile lyases (HNL's) belonging to the α/ß-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/ß-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/ß-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol·M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.


Assuntos
Aldeído Liases/química , Aldeído Liases/genética , Domínio Catalítico , Cristalografia por Raios X/métodos , Esterases/química , Esterases/genética , Hevea/genética , Hevea/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida/métodos , Proteínas de Plantas/genética , Dobramento de Proteína , Especificidade por Substrato , Nicotiana/genética , Nicotiana/metabolismo
19.
Chembiochem ; 10(13): 2213-22, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19672908

RESUMO

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N-alkyl aziridine-2-carboxylates in tBuOH saturated with ammonia and yielded the (2S)-aziridine-2-carboxamide and unreacted (2R)-aziridine-2-carboxylate. Varying the N-1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1'R)-1-phenylethyl substituent reacted approximately ten times faster than substrates with a (1'S)-1-phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5-7) while substrates with either a (1'R)- or (1'S)-1-phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity-binding of the slow enantiomer by an exchange-of-substituents orientation-could not account for the experimental results. However, modeling an umbrella-like-inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1'S)-1-phenylethyl substituent and Thr138 and Ile189 in the acyl-binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella-like-inversion orientation can contribute to enantioselectivity in lipases.


Assuntos
Aziridinas/metabolismo , Candida/enzimologia , Lipase/metabolismo , Aziridinas/química , Catálise , Domínio Catalítico , Proteínas Fúngicas , Ligação de Hidrogênio , Estereoisomerismo
20.
Chemistry ; 15(6): 1370-6, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19115310

RESUMO

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions.


Assuntos
Alcenos/metabolismo , Anidrases Carbônicas/metabolismo , Ródio/química , Alcenos/química , Biocatálise , Anidrases Carbônicas/química , Simulação por Computador , Diálise , Hidrogenação , Isomerismo , Modelos Químicos , Oxirredutases/química , Oxirredutases/metabolismo , Propriedades de Superfície , Zinco/química
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