earticle

영어

Naturally existing functional modules are considered as building blocks for complex structures, and novel functions result from the assembly of different combinations of these building blocks. The identification and characterization of these functional building blocks is relevant to the elucidation of questions regarding protein evolution, folding, and protein engineering for tailored functions. In this study two pyridoxal-5’-phosphate enzymes, Damino acid aminotransferase (D-AAT) and β-tyrosinase (TPL), were characterized about the functional modality after engineered for the higher activity and stability. In D-AAT, a recruitment of the catalytic loop structure (LRcD) from a highly active homolog distinctively changed a weakly active Geobacilli D-AAT: a 68% increase in catalytic activity. Homology modeling suggested that the two tyrosine residues in the EYcY sequence from the Geobacillus enzyme had a π/π-interaction that was replaceable with the salt-bridge interaction between the arginine and aspartate residues in the LRcD sequence.
TPL catalyzes α,β-elimination and β-replacement of L-tyrosine and its related amino acids, with pyridoxal 5'-phosphate (PLP) as the cofactor. Random mutagenesis of the TPL gene resulted in the generation of mutations on the N-terminal arms, which exhibited a higher stability and activity towards L-DOPA as the substrate. The N-terminal arm of the TPL structure was intertwined and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers. When considering the common structural features of α-family PLP enzymes, the N-terminal arm may have increased the rigidity of the cofactor binding architecture of C. freundii TPL through adjusting the quaternary interfaces. Therefore, an enzyme with improved N-terminal arm could be used for the development of a new bioconversion strategy for the efficient production of L-DOPA at high temperatures, where it can catalyze the reaction more actively.
Ideally, the ultimate goal of protein engineering is to create an enzyme for any given chemical reaction. However, our current knowledge of proteins is far from ideal for de novo design of protein function. To overcome this obstacle, two alternate solutions have been extensively pursued: directed evolution based on high throughput screening of mutant library and rational design based on molecular modeling of protein structure. Although evolutionary technologies have accomplished fabulous contribution in biocatalyst developments, it has been compromised by the limited coverage of genetic diversity. Thus, finding the modularity in proteins and limiting the genetic diversity on a particular module could reliably help to combine these two strategies to be most successful and provide further guidance for the improvement of biocatalyst function.