The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine–glyoxylate aminotransferase

Abstract

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

“…The equivalent version of pseudophosphorylated recombinant double mutant CIPK24/SOS2 Thr168Asp Ser228Asp, which also mimics a self-phosphorylated protein at Ser228, severely aggregated and did not crystallize under any tested condition. The application of the consensus-based approach to introduce stabilizing mutations (35,36) was successful. Crystals of the CIPK24/SOS2 variant Glu107Lys/Ser109Asp/ Cys127Ser/Pro81Lys/Leu266Lys (herein after CIPK24/SOS2ΔC T168D) were obtained and the structure was resolved at 3.4 Å resolution (Fig.…”

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications

“…Interestingly each individual variant has either no effect on or reduces the thermal stability, thus demonstrating that the variants have synergistic effects on the protein's structure. These results are in contrast to those obtained with other proteins, in which the effect of consensus mutations’ effects on the stability and activity are often shown to be additive (i.e., single mutants have an effect, and the overall effect in a multiple mutant is essentially additive) …”

“…The lack of additivity in the variants was surprising when compared to previous studies on consensus variants of other enzymes (e.g., refs. , ). This currently remains unexplained, but we hypothesise that the high sensitivity of human galactokinase to variation might be part of the cause.…”

Galactokinase promiscuity: a question of flexibility?

“…Paradoxically, it may be necessary to simultaneously increase the flexibility of the active site, while increasing the global, overall stability of the protein. In pursuit of the latter goal, consensus methods may be a viable method to achieve increased stability without loss of activity [71][72][73]. Overall, it is clear that enzyme engineering methods which rely on altering structure may ultimately be limited; more successful outcomes may be achieved where local and global flexibilities are also considered.…”