Extending enzyme molecular recognition with an expanded amino acid alphabet.

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties.

Aldolase-catalysed stereoselective synthesis of fluorinated small molecules.

The introduction of fluorine has been widely exploited to tune the biological functions of small molecules. Indeed, around 20% of leading drugs contain at least one fluorine atom. Yet, despite profound effects of fluorination on conformation, there is only a limited toolkit of reactions that enable stereoselective synthesis of fluorinated compounds. Aldolases are useful catalysts for the stereoselective synthesis of bioactive small molecules; however, despite fluoropyruvate being a viable nucleophile for some aldolases, the potential of aldolases to control the formation of fluorine-bearing stereocentres has largely been untapped. Very recently, it has been shown that aldolase-catalysed stereoselective carboncarbon bond formation with fluoropyruvate as nucleophile enable the synthesis of many α-fluoro ß-hydroxy carboxyl derivatives. Furthermore, an understanding of the structural basis for the stereocontrol observed in these reactions is beginning to emerge. Here, we review the application of aldolase catalysis in the stereocontrolled synthesis of chiral fluorinated small molecules, and highlight likely areas for future developments.

Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity.

The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90 : 10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30 : 70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation.

Engineering aldolases as biocatalysts.

Aldolases are seen as an attractive route to the production of biologically important compounds due to their ability to form carbon-carbon bonds. However, for many industrial reactions there are no naturally occurring enzymes, and so many different engineering approaches have been used to address this problem. Engineering methods have been used to alter the stability, substrate specificity and stereospecificity of aldolases to produce excellent enzymes for biocatalytic processes. Recently greater understanding of the aldolase mechanism has allowed many successes with both rational engineering approaches and computational design of aldolases. Rational engineering approaches have produced desired enzymes quickly and efficiently while combination of computational design with laboratory methods has created enzymes with activity approaching that of natural enzymes.

Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.