Alanine 101 and alanine 110 of the alpha subunit of <i>Pseudomonas stutzeri</i> OX1 toluene‐<i>o</i>‐xylene monooxygenase influence the regiospecific oxidation of aromatics

Vardar, Gönül; Tao, Yifan; Lee, Jintae; Wood, Thomas K.

doi:10.1002/bit.20637

Cited by 7 publications

(4 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Structures of resting ToMOH and later resting T4moH revealed a contiguous ∼30 Å tunnel extending from the surface into the active site, along with both inner and intermediate chambers along the tunnel (Figure ). Mutagenesis studies with ToMOH supported the viability of its main tunnel as a catalytically relevant path for hydrocarbons, as does the crystallographic evidence showing occupation by relevant aromatic molecules. The open access of the main tunnel in T4moH crystals allowed BML to enter the active site of these crystals, while in contrast, structures of uncomplexed ToMOH and MmoH did not show aromatic molecules in the active site. , T4moD binding closed the main access tunnel in T4moH, and consequently, efforts to soak preformed T4moHD crystals with different aromatic compounds failed to yield structures with these products in the active site even if these compounds were readily entrained elsewhere in the protein.…”

Section: Discussionmentioning

confidence: 71%

“…T4MO oxidizes the natural substrate toluene with ∼97% regiospecificity for the para position . Previous studies with T4MO and related diiron enzymes have shown that mutagenesis can alter the regiospecificity of the enzyme for reaction with toluene and other nonphysiological substrates, ,− in some cases with no deleterious changes in the catalytic properties of the purified enzymes ,, and in others with a broadened range of substrates reacted. ,− For example, G103L T4moH oxidizes toluene with ∼55% regiospecificity for the ortho position, a dramatic change from the natural enzyme, while also retaining equivalent kinetic parameters and coupling …”

mentioning

confidence: 99%

See 1 more Smart Citation

Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

et al. 2012

View full text Add to dashboard Cite

Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric μ-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH(2)-benzoate and p-Br-benzoate showed a μ-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a π-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 Å) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release.

show abstract

Section: Discussionmentioning

confidence: 71%

mentioning

confidence: 99%

Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

et al. 2012

View full text Add to dashboard Cite

show abstract

“…The biocatalyst toluene‐ o ‐xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 (Cafaro et al, ; Radice et al, ) belongs to a remarkable family of bacterial multicomponent monooxygenases (BMM; Notomista et al, ) and has been shown to have a great potential for biotechnological and environmental applications (Notomista et al, ; Vardar and Wood, ; Vardar et al, ,).…”

Section: Introductionmentioning

confidence: 99%

“…Most of these pocket residues have been the subject of several protein engineering studies and shown to influence the catalytic activity and regiospecificity (Cafaro et al, ; Fishman et al, ; McClay et al, ; Notomista et al, ; Pikus et al, ; Rui et al, ; Steffan and McClay, ; Vardar and Wood, ; Vardar et al, ). We have also previously published a summary table showing the important roles of some of these residues on catalysis (Vardar et al, ). Of these residues, F176 and F196 are especially noteworthy since these positions have not been extensively studied through saturation mutagenesis and there are only a few studies performed by rational design approach of site‐directed mutagenesis.…”

Section: Introductionmentioning

confidence: 99%

The role of substrate binding pocket residues phenylalanine 176 and phenylalanine 196 on Pseudomonas sp. OX1 toluene o‐xylene monooxygenase activity and regiospecificity

Sönmez

Yanık-Yıldırım

Wood

et al. 2014

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Saturation mutagenesis was used to generate eleven substitutions of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions F176 and F196 among which nine were novel: F176H, F176N, F176S, F176T, F196A, F196L, F196T, F196Y, F196H, F196I, and F196V. By testing the substrates phenol, toluene, and naphthalene, these positions were found to influence ToMO oxidation activity and regiospecificity. Specifically, TouA variant F176H was identified that had 4.7-, 4.3-, and 1.8-fold faster hydroxylation activity towards phenol, toluene, and naphthalene, respectively, compared to native ToMO. The F176H variant also produced the novel product hydroquinone (61%) from phenol, made twofold more 2-naphthol from naphthalene (34% vs. 16% by the wild-type ToMO), and had the regiospecificity of toluene changed from 51% to 73% p-cresol. The TouA F176N variant had the most para-hydroxylation capability, forming p-cresol (92%) from toluene and hydroquinone (82%) from phenol as the major product, whereas native ToMO formed 30% o-cresol, 19% m-cresol, and 51% of p-cresol from toluene and 100% catechol from phenol. For naphthalene oxidation, TouA variant F176S exhibited the largest shift in the product distribution by producing threefold more 2-naphthol. Among the other F196 variants, F196L produced catechol from phenol two times faster than the wild-type enzyme. The TouA F196I variant produced twofold less o-cresol and 19% more p-cresol from toluene, and the TouA F196A variant produced 62% more 2-naphthol from naphthalene compared to wild-type ToMO. Both of these positions have never been studied through the saturation mutagenesis and some of the best substitutions uncovered here have never been predicted and characterized for aromatics hydroxylation.

show abstract

Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics

Yanık-Yıldırım

Vardar-Schara

2014

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67 ± 0.10 nmol Cl(-)/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively.

show abstract

Alanine 101 and alanine 110 of the alpha subunit of Pseudomonas stutzeri OX1 toluene‐o‐xylene monooxygenase influence the regiospecific oxidation of aromatics

Cited by 7 publications

References 31 publications

Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

The role of substrate binding pocket residues phenylalanine 176 and phenylalanine 196 on Pseudomonas sp. OX1 toluene o‐xylene monooxygenase activity and regiospecificity

Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics

Contact Info

Product

Resources

About