Computational Tools for Rational Protein Engineering of Aldolases

Abstract

In this mini-review we describe the different strategies for rational protein engineering and summarize the computational tools available. Computational tools can either be used to design focused libraries, to predict sequence-function relationships or for structure-based molecular modelling. This also includes de novo design of enzymes. Examples for protein engineering of aldolases and transaldolases are given in the second part of the mini-review.

Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities

“…The limitation of rigid donor specificity was recently overcome by isolation of natural TAs with broad donor specificity l TA from Aeromonas jandaei and d TA from Pseudomonas sp., which accept alanine and serine as donors; thus, l - and d -α-quaternary-α-amino acids were produced (Fesko et al 2010 ). In order to improve the stereoselectivity and substrate specificity of TAs, directed evolution techniques as well as search for promiscuous activities could be promising strategies (Wildmann et al 2012 ; Davids et al 2013 ). This review briefly summarizes the screening and selection methods used in search of new threonine aldolases as well as new structural-functional knowledge which might be helpful for the design of new activities.…”

Engineering aldolases as biocatalysts

“…Much progress has been made towards altering existing enzymes for tailored, stereochemically controlled aldol condensations using a combination of protein engineering strategies. An increasingly common feature of such experiments is the combination of engineering and/or directed evolution with structural modelling, and computational strategies [ 7,12 ]. An ultimate example is the de novo computational design of an aldolase [ 13 ].…”

“…These properties make aldolase-catalysed routes attractive for the production of biologically significant compounds, as these tend to contain multiple functional groups and are often water-soluble making conventional synthetic routes more difficult [ 5 ]. However, naturally occurring aldolases do not exist for many industrially important reactions and protein engineering, directed evolution and de novo enzyme design [ 6–10 ] have all been used to alter properties such as stability, substrate specificity and stereoselectivity to produce tailor made aldolases for use as biocatalysts. Since we reviewed this area in 2008 [ 11 ] it is pleasing to see an increasing use of protein engineering to manipulate aldolases as new biocatalysts, both in their own right and as part of chemical cascade reactions leading to important products (see Table 1 for a summary of recent examples of engineering aldolases).…”

Expanding the threonine aldolase toolbox for the asymmetric synthesis of tertiary α-amino acids

“…In general, aldolases can convert a variety of acceptor substrates, whereas the donor components are confined, thus narrowing the possible product range (Fessner 2011). Protein engineering techniques, screening for novel aldolase activities in nature and de novo design of aldolases are currently methods of choice to enable novel synthetic applications of aldolases (Baker and Seah 2012;Wildmann et al 2012;Windle et al 2014). Several attempts to change the donor specificity of aldolases have been performed by directed evolution approaches; however, only small structural, isosteric modification of the donor component could be implemented (Müller 2012;Fesko and Gruber 2013).…”