Insights into the substrate selectivity of Bambusa oldhamii phenylalanine ammonia-lyase 1 and 2 through mutational analysis

Phenylalanine ammonia-lyase (PAL) links the plant primary and secondary metabolisms, and its product, trans-cinnamic acid, is derived into thousands of diverse phenylpropanoids. Bambusa oldhamii BoPAL4 has broad substrate specificity using L-phenylalanine, L-tyrosine, and L-3,4-dihydroxy phenylalanine (L-DOPA) as substrates to yield trans-cinnamic acid, p-coumaric acid, and caffeic acid, respectively. The optimum reaction pH of BoPAL4 for three substrates was measured at 9.0, 8.5, and 9.0, respectively. The optimum reaction temperatures of BoPAL4 for three substrates were obtained at 50, 60, and 40 °C, respectively. The Km values of BoPAL4 for three substrates were 2084, 98, and 956 μM, respectively. The kcat values of BoPAL4 for three substrates were 1.44, 0.18, and 0.06 σ-1, respectively. The major substrate specificity site mutant, BoPAL4-H123F, showed better affinity toward L-phenylalanine by decreasing its Km value to 640 μM and increasing its kcat value to 1.87 s-1. In comparison to wild-type BoPAL4, the specific activities of BoPAL4-H123F using L-tyrosine and L-DOPA as substrates retained 5.4% and 17.8% residual activities. Therefore, L-phenylalanine, L-tyrosine, and L-DOPA are bona fide substrates for BoPAL4.

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Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From Thermobifida fusca YX

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.615614 ◽

2021 ◽

Vol 7 ◽

Author(s):

Kiran-Kumar Shivaiah ◽

Bryon Upton ◽

Basil J. Nikolau

Keyword(s):

Substrate Specificity ◽

Quaternary Structure ◽

Mutational Analysis ◽

Streptomyces Coelicolor ◽

Thermobifida Fusca ◽

Substrate Selectivity ◽

Acetyl Coa ◽

Catalytic Subunits ◽

Chain Acyl

Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.

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Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris

Protein Expression and Purification ◽

10.1016/j.pep.2010.01.009 ◽

2010 ◽

Vol 71 (2) ◽

pp. 224-230 ◽

Cited By ~ 26

Author(s):

Lu-Sheng Hsieh ◽

Chuan-Shan Yeh ◽

Hung-Chi Pan ◽

Chieh-Yang Cheng ◽

Chien-Chih Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Pichia Pastoris ◽

Phenylalanine Ammonia Lyase ◽

Cloning And Expression ◽

Bambusa Oldhamii ◽

Phenylalanine Ammonia Lyase Gene

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Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H + transporters in fungi and plants

Open Biology ◽

10.1098/rsob.140070 ◽

2014 ◽

Vol 4 (6) ◽

pp. 140070 ◽

Cited By ~ 10

Author(s):

Manuel Sanguinetti ◽

Sotiris Amillis ◽

Sergio Pantano ◽

Claudio Scazzocchio ◽

Ana Ramón

Keyword(s):

Amino Acids ◽

Aspergillus Nidulans ◽

Vibrio Parahaemolyticus ◽

Mutational Analysis ◽

Three Dimensional ◽

Random Mutagenesis ◽

Membrane Transporters ◽

Substrate Selectivity ◽

Dimensional Model ◽

Three Dimensional Model

We present the first account of the structure–function relationships of a protein of the subfamily of urea/H + membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.

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