A novel genetic selection system for PLP-dependent threonine aldolases

被引:21
作者
Giger, Lars [1 ]
Toscano, Miguel D. [1 ,2 ]
Bouzon, Madeleine [3 ]
Marliere, Philippe [4 ]
Hilvert, Donald [1 ]
机构
[1] ETH, Organ Chem Lab, CH-8093 Zurich, Switzerland
[2] Novozymes AS, DK-2880 Bagsvaerd, Denmark
[3] IG, DSV, CEA, F-91057 Evry, France
[4] Heurisko USA Inc, Barksdale Profess Ctr 113, Newark, DE USA
关键词
Genetic complementation; Pyridoxal-5 '-phosphate; Aldolase; Combinatorial mutagenesis; Catalysis; Mechanism; Directed evolution; ALPHA-AMINO ACIDS; ACTIVE-SITE; SERINE HYDROXYMETHYLTRANSFERASE; CHORISMATE MUTASE; CATALYTIC TRIAD; ENZYMES; MUTAGENESIS; GLYCINE; BIOSYNTHESIS; PURIFICATION;
D O I
10.1016/j.tet.2012.05.097
中图分类号
O62 [有机化学];
学科分类号
070303 ; 081704 ;
摘要
Threonine aldolases are versatile pyridoxal-5'-phosphate (PLP)-dependent enzymes key to glycine, serine and threonine metabolism. Because they catalyze the reversible addition of glycine to an aldehyde to give beta-hydroxy-alpha-amino acids, they are also attractive as biotechnological catalysts for the diastereo-selective synthesis of many pharmaceutically useful compounds. To study and evolve such enzymes, we have developed a simple selection system based on the simultaneous inactivation of four genes involved in glycine biosynthesis in Escherichia coli. Glycine prototrophy in the deletion strain is restored by expression of a gene encoding an aldolase that converts beta-hydroxy-alpha-amino acids, provided in the medium, to glycine and the corresponding aldehyde. Combinatorial mutagenesis and selection experiments with a previously uncharacterized L-threonine aldolase from Caulobacter crescentus CB15 (Cc-LTA) illustrate the power of this system. The codons for four active site residues, His91, Asp95, Glu96, and Asp176, were simultaneously randomized and active variants selected. The results show that only His91, which it-stacks against the PLP cofactor and probably serves as the catalytic base in the carbon-carbon bond cleavage step, is absolutely required for aldolase activity. In contrast, Asp176, one of the most conserved residues in this enzyme superfamily, can be replaced conservatively by glutamate, albeit with a >5000-fold decrease in efficiency. Though neither Asp95 nor Glu96 is catalytically essential, they appear to modulate substrate binding and His91 activity, respectively. The broad dynamic range of this novel selection system should make it useful for mechanistic investigations and directed evolution of many natural and artificial aldolases. (C) 2012 Elsevier Ltd. All rights reserved.
引用
收藏
页码:7549 / 7557
页数:9
相关论文
共 55 条
  • [11] L-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase - A subgroup of strictly related enzymes specialized for different functions
    Contestabile, R
    Paiardini, A
    Pascarella, S
    di Salvo, ML
    D'Aguanno, S
    Bossa, F
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 2001, 268 (24): : 6508 - 6525
  • [12] THE CATALYTIC ROLE OF THE ACTIVE-SITE ASPARTIC-ACID IN SERINE PROTEASES
    CRAIK, CS
    ROCZNIAK, S
    LARGMAN, C
    RUTTER, WJ
    [J]. SCIENCE, 1987, 237 (4817) : 909 - 913
  • [13] One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
    Datsenko, KA
    Wanner, BL
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (12) : 6640 - 6645
  • [14] Threonine aldolases-screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids
    Dueckers, Nina
    Baer, Katrin
    Simon, Sabine
    Groeger, Harald
    Hummel, Werner
    [J]. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2010, 88 (02) : 409 - 424
  • [15] Pyridoxal phosphate enzymes: Mechanistic, structural, and evolutionary considerations
    Eliot, AC
    Kirsch, JF
    [J]. ANNUAL REVIEW OF BIOCHEMISTRY, 2004, 73 : 383 - 415
  • [16] Four types of threonine aldolases:: Similarities and differences in kinetics/thermodynamics
    Fesko, Kateryna
    Reisinger, Christoph
    Steinreiber, Johannes
    Weber, Hansjoerg
    Schuermann, Martin
    Griengl, Herfried
    [J]. JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 2008, 52-53 : 19 - 26
  • [17] Biocatalytic Access to α,α-Dialkyl-α-amino Acids by a Mechanism-Based Approach
    Fesko, Kateryna
    Uhl, Michael
    Steinreiber, Johannes
    Gruber, Karl
    Griengl, Herfried
    [J]. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2010, 49 (01) : 121 - 124
  • [18] DERIVATION OF GLYCINE FROM THREONINE IN ESCHERICHIA-COLI K-12 MUTANTS
    FRASER, J
    NEWMAN, EB
    [J]. JOURNAL OF BACTERIOLOGY, 1975, 122 (03) : 810 - 817
  • [19] Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy
    Gamper, M
    Hilvert, D
    Kast, P
    [J]. BIOCHEMISTRY, 2000, 39 (46) : 14087 - 14094
  • [20] Diastereoselective synthesis of l-threo-3,4-dihydroxyphenylserine by low-specific l-threonine aldolase mutants
    Gwon, Hui-Jeong
    Baik, Sang-Ho
    [J]. BIOTECHNOLOGY LETTERS, 2010, 32 (01) : 143 - 149