Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase

A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

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Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase

Blood ◽

10.1182/blood.v81.3.808.808 ◽

1993 ◽

Vol 81 (3) ◽

pp. 808-814 ◽

Cited By ~ 1

Author(s):

T Nagai ◽

K Shirabe ◽

T Yubisui ◽

M Takeshita

Keyword(s):

Enzyme Activity ◽

Cytochrome B5 ◽

Base Change ◽

Mutant Enzyme ◽

Type I ◽

Wild Type ◽

Cytochrome B5 Reductase ◽

Type Iii ◽

Patient Reported ◽

Nadh Cytochrome

Abstract A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

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A Novel Heterologous Expression System for Characterization of Individual NADH-Cytochrome b5 Reductase Variants in Recessive Congenital Methemoglobinemia.

Blood ◽

10.1182/blood.v106.11.1676.1676 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1676-1676

Author(s):

Melanie J. Percy ◽

L. J. Crowley ◽

C. A. Davis ◽

J. Boudreaux ◽

D. M. Layton ◽

...

Keyword(s):

Thermal Stability ◽

Enzyme Activity ◽

Heterologous Expression ◽

Expression System ◽

Cytochrome B5 ◽

Type I ◽

Type Ii ◽

Wild Type ◽

Heterologous Expression System ◽

Nadh Cytochrome

Abstract Deficiency of NADH-cytochrome b5 reductase (cb5r) causes two clinically distinct phenotypes of recessive congenital methemoglobinemia (RCM). Type I patients often manifest cyanosis from birth, and in type II patients the cyanosis is accompanied by severe neurological impairment. The mechanisms responsible for the phenotypic differences between the two subgroups remain to be defined. The majority of patients harbor two different mutant alleles. To date 39 mutant variants of cb5r have been identified, 2 of which are common to both types of RCM. In order to characterize the individual cb5r variant proteins we have developed a novel heterologous expression system based on the structures of the rat and human proteins derived by X-ray crystallography. The system permits the investigation of the catalytic efficiencies, protein thermostability, FAD cofactor properties and substrate (NADH/NAD+) affinities of the variants. We have investigated four patients with type I RCM, one of whom was homozygous for the D239G mutation. The other three were compound heterozygous: R159-/D239G; G75S/V252M; and P275L/G291D, and one mutation, P275L, was novel. All patients showed reduced enzyme activity, in the range 0.5 to 5.8 IU/g Hb compared to normal values of 7.2 to 26.9 IU/g Hb. Individual variant proteins were prepared and the analytical data are summarised in the Table below. Variant Catalytic Efficiency (% of normal) Thermal Stability (T50°C) NADH affinity (Km) NAD+ affinity (Ks) ND - not determined G75S 11 48 Normal 9-fold ↑ R159- 0 ND ND ND D239G 2 56 40-fold ↓ ND V252M 9 53 9-fold ↓ 18-fold ↑ E255- 0.4 51 100-fold ↓ ND P275L 0.2 53 437-fold ↓ ND G291D 43 49 1.3-fold ↓ 1.1-fold ↑ Wild type 100 57 normal normal As expected all of the variants generated had decreased enzyme activity compared to wild type heterologous protein, supporting the validity of this approach. Thermal stability was decreased in the G75S, V252M and G291D variants. G75 is present in a highly conserved region in the FAD-binding lobe. Although it does not interact directly with the FAD prosthetic group it is important for association with cytochrome b5. Substitution of glycine at residue 75 by serine resulted in decreased enzyme activity and stability, with a marginal decrease in NADH affinity. The R159- variant protein was unstable and could not be isolated. Both the D239G and P275L mutations significantly reduced the affinity of cb5r for NADH, by 40-fold and 437-fold respectively. The rat cb5r model suggests that residue D239 is key for selecting between the NADPH and NADH pyridine nucleotides. This was confirmed by the 40-fold decrease in affinity for NADH and a 125-fold increase in affinity for NADPH. Residue P275 is located in a highly conserved region, which is important for the correct positioning and binding of NADH. Consequently, substitution of proline at 275 would affect the affinity of cb5r towards NADH, which was confirmed by the affinity constant measurements. These studies provide important information about the structure-function relationships of the variant cb5r proteins which may impart useful insights into the pathophysiological differences between type I and type II RCM.

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A novel mutation in the NADH-cytochrome b5 reductase gene of a Chinese patient with recessive congenital methemoglobinemia

Blood ◽

10.1182/blood.v95.10.3250 ◽

2000 ◽

Vol 95 (10) ◽

pp. 3250-3255 ◽

Cited By ~ 21

Author(s):

Yao Wang ◽

Yu-Shui Wu ◽

Pei-Zhen Zheng ◽

Wen-Xi Yang ◽

Guo-An Fang ◽

...

Keyword(s):

Clinical Symptoms ◽

Novel Mutation ◽

Cytochrome B5 ◽

Clinical Manifestations ◽

Mutant Enzyme ◽

Chinese Family ◽

Coding Region ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

Abstract Recessive congenital methemoglobinemia due to nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is classified into 2 clinical types: type 1 (erythrocyte type) and type 2 (generalized type). We found a Chinese family with type 1 recessive congenital methemoglobinemia, the patients from which were diagnosed according to clinical symptoms and b5R enzyme activity in the blood cells. To learn the molecular basis of type 1 recessive congenital methemoglobinemia in this Chinese family, we isolated total RNA from the peripheral leukocytes of the propositus and b5R complementary DNA (cDNA) by reverse transcription– polymerase chain reaction (RT-PCR). The coding region of the b5R cDNA was analyzed by sequencing the cloned PCR products. The results showed that the propositus was homozygous for a G→A transition at codon 203 in exon 7, changing a cysteine to a tyrosine (Cys203Tyr). To characterize the mutant enzyme, both glutathione S-transferase (GST)-fused wild-type b5R and GST-fused mutant Cys203Tyr b5R were expressed in Escherichia coli and affinity purified. The results showed that the catalytic activity of the enzyme was not much affected by this amino acid substitution, but the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin. These properties of the mutant enzyme would account for the restricted b5R deficiency and mild clinical manifestations of these type 1 patients. The finding of this novel mutation makes codon 203 the only position within the b5R gene at which more than 1 mutation has been found.

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Three Novel Mutations in CYB5R3 Gene Causing NADH-cytochrome b5 Reductase Enzyme Deficiency Leads to Recessive Congenital Methaemoglobinemia

10.21203/rs.3.rs-732442/v1 ◽

2021 ◽

Author(s):

Anuradha Deorukhkar ◽

Anuja KULKARNI ◽

Prabhakar S Kedar

Keyword(s):

Splice Site ◽

Genetic Disorders ◽

Cytochrome B5 ◽

Neurological Impairment ◽

Enzyme Deficiency ◽

Type I ◽

Cytochrome B5 Reductase ◽

Pathogenic Variants ◽

Nucleotide Insertion ◽

Nadh Cytochrome

Abstract Two types of recessive congenital methaemoglobinemia (RCM) is caused by NADH-dependent cytochrome b5 reductase enzyme deficiency encoded by CYB5R3 gene. RCM-I is characterized by higher methaemoglobin levels (>2 g/dL), causing only cyanosis, whereas RCMR-II is associated with cyanosis with neurological impairment. The present study discovered three novel homozygous pathogenic variants of CYB5R3 causing RCM I and II in four unrelated Indian patients. In patient-1 and patient-2 of are of RCM type I caused due to novel c.175C>T (p.Arg59Cys) and other reported c.469T>C (p.Phe157Ser) missense pathogenic variants respectively, whereas patient-3 and patient-4 presented with the RCM type II are related to developmental delay with cyanosis since birth due to a novel homozygous (g.25679_25679delA) splice-site deletion and novel homozygous c.824_825insC (p.Pro278ThrfsTer367) single nucleotide insertion. The CYB5R3 transcript levels were estimated by qRT-PCR in the splice-site deletion, which was 0.33fold of normal healthy control. The insertion of nucleotide C resulted in a frame-shift of termination codon are associated with neurological impairment. This study can help to conduct genetic counselling and, subsequently, prenatal diagnosis of high-risk genetic disorders.

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Enzymatic instability of NADH-cytochrome b5 reductase as a cause of hereditary methemoglobinemia type I (red cell type).

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)88718-5 ◽

1992 ◽

Vol 267 (28) ◽

pp. 20416-20421

Author(s):

K Shirabe ◽

T Yubisui ◽

N Borgese ◽

C.Y. Tang ◽

D.E. Hultquist ◽

...

Keyword(s):

Cytochrome B5 ◽

Type I ◽

Red Cell ◽

Cell Type ◽

Cytochrome B5 Reductase ◽

Nadh Cytochrome

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A novel G143D mutation in the NADH-cytochrome b5 reductase gene in an Indian patient with type I recessive hereditary methemoglobinemia

Blood Cells Molecules and Diseases ◽

10.1016/j.bcmd.2007.09.006 ◽

2008 ◽

Vol 40 (3) ◽

pp. 323-327 ◽

Cited By ~ 5

Author(s):

Prabhakar S. Kedar ◽

Prashant Warang ◽

Anita H. Nadkarni ◽

Roshan B. Colah ◽

Kanjaksha Ghosh

Keyword(s):

Cytochrome B5 ◽

Type I ◽

Indian Patient ◽

Cytochrome B5 Reductase ◽

Reductase Gene ◽

Nadh Cytochrome

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NADH-cytochrome b5 reductase in a Turkish family with recessive congenital methaemoglobinaemia type I: Table 1

Journal of Clinical Pathology ◽

10.1136/jcp.2008.058701 ◽

2008 ◽

Vol 61 (10) ◽

pp. 1122-1123 ◽

Cited By ~ 7

Author(s):

M J Percy ◽

D Aslan

Keyword(s):

Cytochrome B5 ◽

Blood Gas Analysis ◽

Gas Analysis ◽

Type I ◽

Missense Mutations ◽

Arterial Blood Gas ◽

Cytochrome B5 Reductase ◽

Arterial Blood ◽

Turkish Family ◽

Nadh Cytochrome

The development of cyanosis at birth, the so-called blue baby syndrome, alerts paediatricians to the presence of congenital heart disease. In rare cases where the arterial blood gas analysis is normal the cyanosis is a consequence of methaemoglobinaemia. There are three distinct origins of methaemoglobinaemia; the presence of a haemoglobin variant, environmental toxicity and deficiency of cytochrome b5 reductase (cb5r). Two children born to two sets of first-degree related parents were cyanotic from birth. Differential diagnosis eliminated cardiac and pulmonary abnormalities. Measurement of methaemoglobin levels confirmed recessive congenital methaemoglobinaemia (RCM) and treatment with ascorbic acid was commenced. In the absence of neurological defects, type I disease was diagnosed. Sequence analysis of CYB5R3 revealed two different missense mutations (one which is novel, Ile85Ser) in the two families. Neither of the mutations was located in the FAD or the NADH binding sites of cb5r, thus supporting a diagnosis of type I disease.

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