Abstract
In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in the diagnosis and treatment of this serious disease.
Highlights
Enzymes dependent on the vitamin B12 coenzymes, adenosyl-B12 (Ado-B12)1 and methyl-B12 (CH3-B12), have a broad but uneven distribution among living forms [1,2,3]
Such deficiencies can result from inborn errors in the methylmalonyl-CoA mutase (MCM) structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM
We propose that the human gene identified here coincides with the cobalamin b (cblB) complementation group, defects in which lead to methylmalonyl aciduria
Summary
Enzymes dependent on the vitamin B12 coenzymes, adenosyl-B12 (Ado-B12) and methyl-B12 (CH3-B12), have a broad but uneven distribution among living forms [1,2,3]. Four complementation groups (cblABCD), associated with methylmalonyl aciduria, are thought to correspond to genes involved in the conversion of X-B12s to Ado-B12. It was found that a plasmid-encoded source of ATR enzyme restored DDH activity to a cobA pduO double mutant [20] Because this enzymatic activity can be readily detected on aldehyde indicator medium, we expected that the ATR-deficient Salmonella strain could be used to screen expression libraries for cDNAs that encode ATR enzymes. The homologous human cDNA and its corresponding gene were identified by sequence similarity searching Both the human and bovine cDNAs are shown to express ATR activity and complement an ATR-deficient bacterial mutant. The identification of genes involved in methylmalonyl aciduria is important for the development of improved methods for the diagnoses and treatment of this devastating disorder
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