To study this mechanism we developed an expression system to produce recombinant CBS mutant proteins identified in a group of 18 CBS deficient patients presenting a disturbed SAM regulation, and localized away from the C-terminal domain, namely in the N-terminal domain (P49L) and in the catalytic core (G151R, G153R, K269del, I278T, R336H). CBS wild-type cDNA was cloned into pET28b expression vector. Mutations were obtained by site-directed mutagenesis. Enzyme activity and SAM activation ratio were determined according to standard procedures. Expression levels and purity grades were evaluated by SDS-PAGE and oligomeric profile by native-gel electrophoresis. Functional CBS wild-type protein was obtained in high yield and purity grade. From the studied CBS mutants only P49L presented a high residual activity (81% ofWT) but a lower SAM activation (20.3% of WT Inhibition of cellular methylation reactions by S-adenosyl homocysteine (AdoHcy), which accumulates in the setting of hyperhomocysteinemia, has been suggested to contribute to endothelial dysfunction in homocysteine (Hcy)-related vascular disease. We aimed at determining whether intracellular AdoHcy accumulation affects NO production by human endothelial cells. Human vascular endothelial cells were incubated with adenosine-2,3dialdehyde (ADA) (0, 5, 10 and 20 μmol/L), an inhibitor of AdoHcy hydrolase, for 12 to 24 hours. Extracellular Hcy and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (eNOS), were measured by HPLC. Intracellular AdoHcy was determined by LC-MS/ MS. Nitrite was quantified by Griess reaction to evaluate NO production. Coincubations with L-NG-nitroarginine (L-NNA) (1 mmol/L), an eNOS inhibitor, were also performed. eNOS transcriptional and translational levels were studied by qRT-PCR and Western blotting, respectively. eNOS function was evaluated by assessing the effectiveness of L-arginine to L-citrulline conversion in cell lysates. ADA elicited intracellular accumulation AdoHcy, while extracellular Hcy, ADMA and nitrite levels decreased. Co-incubation with L-NNA suppressed this effect. For ADA at 20 μmol/L, eNOS expression and activity decreased by 40% and 20%, respectively. Intriguingly, transcription of NOS3 increased. Thus, ADMA-independent postranscriptional mechanisms modulated by cellular methylation capacity may lower NO production by vascular endothelial cells. Mutations in the gene encoding methionine synthase result in methylcobalamin deficiency G (CblG) disorder leading to hyperhomocysteinemia and low methionine. The most frequent clinical findings reported are megaloblastic anemia and neurological symptoms. Patient 1 was hypotonic at birth and anaemia was observed at one month. She was weak, had feeding difficulties and a gastrostomy was installed at 7 months. Delayed development led to diagnosis and treatment start at one year. Almost 6 years old she presented with kidney failure due to an atypical hemolytic uraemic syndrome (HUS) from which she recovered after 6 months. Now at age eleven she has short stature and attends a special school. Patient 2 presented neonatally with apnoeas, hypotonia, seizures and poor weight gain. After initiation of treatment he recovered but not to complete normality. At two years he also developed an atypical HUS and a tracheal stenosis was diagnosed. Now, 3 years old, kidney function has normalized but he still suffers from hypertension, needs both a gastrostomy and a tracheostomy tube and has a short stature. His cognitive function is almost normal.