Background: Congenital adrenal hyperplasia is caused by insufficient adrenal steroid biosynthesis due to impaired steroidogenic enzymes. The majority of patients suffers from deficiency of 21-hydroxylase (CYP21) coded by the CYP21A2 gene.

Objective: To study the functional and structural consequences of the novel CYP21A2 missense mutation c.364A>C (K121Q) detected in a female patient with nonclassical 21-hydroxylase deficiency. The patient was compound heterozygous for the novel K121Q mutation and the mild P453S mutation.

Results: In vitro expression analysis of the mutant K121Q enzyme in transiently transfected COS-7 cells revealed reduced CYP21 activity of 14.0 ± 5% for the conversion of 17-hydroxyprogesterone and 19.5 ± 4% for the conversion of progesterone. K121 is located on helix C in the CYP21 protein, which is part of the heme coordinating system. In addition helix C is involved in the interaction with the electron providing enzyme P450 oxidoreductase. Protein modelling revealed that the substitution of glutamine for the basic amino acid lysine introduces an electrostatic change on the surface of CYP21 and may additionally change heme coordination. We hypothesize that the electron flux between POR and CYP21 is impaired and, moreover, substrate affinity is altered due to heme dislocation with K121Q.

Conclusion: Both, the interaction of POR and CYP21 as well as heme coordination are likely to be disturbed due to the K121Q mutation. Our data exemplify how the combination of in vitro expression and structural protein analysis provide novel insights into molecular mechanisms of reduced CYP21 activity eventually explaining the patient's phenotype.