Functional Characterization of the Vitamin K2 Biosynthetic Enzyme UBIAD1

Abstract

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.

Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi

“…Membrane-associated Sar1 subsequently recruits the COPII dimers, Sec23/24 and Sec13/31, resulting in budding of vesicles from ER human UBIAD1 participates in coordination of Mg 2+ ions that mediate interactions with the pyrophosphate group. The SCD-associated N102S mutation in UBIAD1 markedly diminishes MK-4 synthetic activity (50) and causes the mutant enzyme to become sequestered in the ER of isoprenoid-replete cells (Fig. 4) (12).…”

Does statins promote vascular calcification in chronic kidney disease?

“…As statin users were older, had higher BMI and had higher burden of diabetes and CVD (Table 3), our immediate reflex was that the observed link is a reflection of 'confounding by indication' [42]. Lipophilic statins inhibit the enzymatic activity of UbiA prenyltransferase domain-containing protein 1 (UBIAD1) and enzyme which plays a significant role in vitamin K2 synthesis [52]. In the present study, the link between statins and increased CAC score remained significant after adjustment for age (also when age was included in the model as a continuous variable), diabetes, BMI and inflammation.…”

“…Vitamin K deficiency is common in ESRD [50], and vitamin K supplementation has been proposed to arrest progression of vascular calcification [51]. Lipophilic statins inhibit the enzymatic activity of UbiA prenyltransferase domain-containing protein 1 (UBIAD1) and enzyme which plays a significant role in vitamin K2 synthesis [52]. Therefore, it is tempting to speculate that statins may accelerate artery calcification [38,39] via depletion of vascular vitamin K2 levels, thereby increasing uncarboxylated matrix gla protein (MGP) [53,54].…”

Schnyder corneal dystrophy-associated UBIAD1 is defective in MK-4 synthesis and resists autophagy-mediated degradation

“…Structural analyses of archaeal UbiA prenyltransferases reveal that many residues corresponding to SCD-associated mutations in human UBIAD1 localize to the active site of the enzyme, indicating SCD-associated UBIAD1 variants are enzymatically defective (9,10). Indeed, several SCD-associated UBIAD1 variants exhibited reduced enzymatic activity (6,11); however, whether other variants of the enzyme are defective in MK-4 synthesis is unknown.…”