The quest for an enzyme with desired property is high for biocatalyic production of valuable products in industrial biotechnology. Synthetic biology and metabolic engineering also increasingly require an enzyme with unusual property in terms of substrate spectrum and catalytic activity for the construction of novel circuits and pathways. Structure-guided enzyme engineering has demonstrated a prominent utility and potential in generating such an enzyme, even though some limitations still remain. In this chapter, we present some issues regarding the implementation of the structural information to enzyme engineering, and exemplify the structure-guided rational approach to the design of an enzyme with desired functionality such as substrate specificity and catalytic efficiency.