HighlightsAldolase enzymes are attractive candidates as biocatalysts.Stability, stereoselectivity and substrate specificity of aldolases have been altered.Aldolases with desirable activities have been produced using a variety of methods.Combining computational with other methods produces efficient designer aldolases.

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 pKa 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.

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.protein engineering | aldolases | chemical modification E nzymes are phenomenally powerful catalysts that increase reaction rates by up to 10 18 -fold (1, 2), and a new era of enzyme applications has been opened by the advancement of protein engineering and directed evolution to provide new, or improved, enzymes for industrial biocatalysis. Enzymes are attractive catalysts because they are highly selective, carrying out regio-, chemo-, and stereoselective reactions that are challenging for conventional chemistry. Moreover, enzymes are efficient catalysts, function under mild conditions with relatively nontoxic reagents, and enable the production of relatively pure products, minimizing waste generation. In recent years, there has been much success in engineering enzymes for desired reactions (3-5) using methods such as rational protein engineering (6-8), directed evolution (9-11), and, most recently, computational enzyme design (12-15).Enzymes found in Nature achieve catalysis using active sites generally composed of only 20 canonical amino acids, which are encoded at the genetic level, plus the rarer selenocysteine and 1-pyrrolysine. However, many enzymes also rely on one or more of 27 small organic cofactor molecules and/or 13 metal ions for their function (16). In addition, in some cases, Nature has exploited noncanonical amino acids (Ncas) in catalysis to extend its catalytic repertoire: for example, the quinones TPQ, LTQ, TTQ, and CTQ, respectively, in ...

The stereochemical course of aldolase-catalysed reaction between fluoropyruvate and aldehydes is described.