Background: Folate-dependent one-carbon metabolism provides one-carbon units for several biological processes.
This pathway is highly compartmentalized in eukaryotes, with the mitochondrial pathway producing formate for
use in cytoplasmic processes. The mitochondrial enzyme MTHFD2 has been reported to use NAD+ as a cofactor
while the isozyme MTHFD2L utilizes NAD+ or NADP+ at physiologically relevant conditions. Because MTHFD2 is
highly expressed in many cancer types, we sought to determine the cofactor preference of this enzyme.
Results: Kinetic analysis shows that purified human MTHFD2 exhibits dual redox cofactor specificity, utilizing either
NADP+ or NAD+ with the more physiologically relevant pentaglutamate folate substrate.
Conclusion: These results show that the mitochondrial folate pathway isozymes MTHFD2 and MTHFD2L both
exhibit dual redox cofactor specificity. Our kinetic analysis clearly supports a role for MTHFD2 in mitochondrial
NADPH production, indicating that this enzyme is likely responsible for mitochondrial production of both NADH
and NADPH in rapidly proliferating cells.

• Human mitochondrial MTHFD2 is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate cyclohydrolase