Exploring the catalytic mechanisms and physiological function of senescence marker protein 30 via crystallography, enzyme kinetics, and isothermal titration calorimetry

Senescence marker protein 30 (SMP30) is a multifunctional protein involved in cellular Ca2+ homeostasis and the biosynthesis of ascorbate in nonprimate mammals. The primary protein structure is highly conserved among vertebrates, suggesting the existence of a significant physiological function common to all mammals. Enzymatic activities of SMP30 include lactone and organophosphate hydrolysis. SMP30 is a six-bladed beta-propeller fold enzyme that shares structural and functional similarity with paraoxonase 1 (PON1), and squid DFPase. The two catalytic mechanisms of SMP30 have not yet been elucidated. Furthermore, the physiological function and natural substrate of human SMP30 is unknown. The work described here explores the catalytic mechanisms of SMP30 and the physiological function of human SMP30. Expression and purification protocols for both human and mouse forms of SMP30 were improved to obtain an improved yield and purity. Comparison of mouse and human forms of SMP30 revealed little difference in catalytic activity or active site structure. We propose that the catalytic activity of human SMP30 is also important for its physiological function, although unknown at this time. Mammalian expression studies revealed that SMP30 is prone to degradation when overexpressed. Based on kinetics experiments and available crystal structures, a physiological substrate of mouse SMP30 in the ascorbate biosynthesis pathway was called into question. We propose that SMP30 may first close substrates into six-membered rather than five-membered lactones. Although the identities of residues necessary for catalysis are known, clear mechanisms have not been established. Kinetic and metal-binding studies of two active site mutants, D204N and E18Q, suggest that these metal-coordinating residues may play a more direct role in the catalytic activities of SMP30. Once believed to be a Ca2+-binding protein, studies have shown that SMP30 has a very low Ca2+-binding affinity. The greatest lactonase activity was observed in the presence of Zn2+. This work describes the determination of the Zn2+-binding constant in the absence of substrate. We propose that Zn2+ may be a physiologically relevant metal ion for SMP30. Based on analysis of high resolution X-ray data, we also suggest that high concentrations of the product, L-gulonate, may affect metal binding by stripping metal from the protein.