Molybdenum Cofactor Catabolism Unravels the Physiological Role of the Drug Metabolizing Enzyme Thiopurine S‐Methyltransferase

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco‐deficiency, its catabolic pathway remains unknown. Here, we identified the urothione‐synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S‐methylating enzyme corresponds to thiopurine S‐methyltransferase (TPMT), a highly polymorphic drug‐metabolizing enzyme associated with drug‐related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild‐type and knock‐out (Tpmt−/−) mice as well as human liver cytosol. Urothione levels were quantified by liquid‐chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT‐genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population‐based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin‐methylating activity was associated with TPMT activity determined with its drug substrate (6‐thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt−/− mice. Urinary urothione concentration in TPMT‐deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population‐based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug‐ and endogenous substrates.

[1]  W. Rathmann,et al.  Cohort Profile Update: The Study of Health in Pomerania (SHIP). , 2022, International journal of epidemiology.

[2]  G. Schwarz,et al.  Molybdenum cofactor biology, evolution and deficiency. , 2020, Biochimica et biophysica acta. Molecular cell research.

[3]  F. Wong,et al.  Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A: a prospective cohort study , 2015, The Lancet.

[4]  Howard L McLeod,et al.  Nomenclature for alleles of the thiopurine methyltransferase gene , 2013, Pharmacogenetics and genomics.

[5]  T E Klein,et al.  Clinical Pharmacogenetics Implementation Consortium Guidelines for Thiopurine Methyltransferase Genotype and Thiopurine Dosing: 2013 Update , 2013, Clinical pharmacology and therapeutics.

[6]  K. Okamoto,et al.  Send Orders of Reprints at Reprints@benthamscience.net Chemical Nature and Reaction Mechanisms of the Molybdenum Cofactor of Xanthine Oxidoreductase , 2022 .

[7]  G. Schwarz,et al.  Metal insertion into the molybdenum cofactor: product-substrate channelling demonstrates the functional origin of domain fusion in gephyrin. , 2013, The Biochemical journal.

[8]  H. Guchelaar,et al.  Pharmacogenetics: From Bench to Byte— An Update of Guidelines , 2011, Clinical pharmacology and therapeutics.

[9]  W. Rathmann,et al.  Cohort profile: the study of health in Pomerania. , 2011, International journal of epidemiology.

[10]  T E Klein,et al.  Pharmacogenetics Implementation consortium Guidelines for Thiopurine Methyltransferase Genotype and Thiopurine Dosing , 2011 .

[11]  F. Wong,et al.  Successful Treatment of Molybdenum Cofactor Deficiency Type A With cPMP , 2010, Pediatrics.

[12]  D. Keppler,et al.  Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver , 2009, Hepatology.

[13]  R. Mendel,et al.  Molybdenum cofactors, enzymes and pathways , 2009, Nature.

[14]  M. Hiratsuka,et al.  Functional characterization of 23 allelic variants of thiopurine S-methyltransferase gene (TPMT*2 – *24) , 2008, Pharmacogenetics and genomics.

[15]  M. Eichelbaum,et al.  Highly multiplexed genotyping of thiopurine s-methyltransferase variants using MALD-TOF mass spectrometry: reliable genotyping in different ethnic groups. , 2008, Clinical chemistry.

[16]  U. Hofmann,et al.  Role of genetic and nongenetic factors for fluorouracil treatment-related severe toxicity: a prospective clinical trial by the German 5-FU Toxicity Study Group. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[17]  M. Relling,et al.  Differential effects of targeted disruption of thiopurine methyltransferase on mercaptopurine and thioguanine pharmacodynamics. , 2007, Cancer research.

[18]  H. Herfarth,et al.  Monitoring of Thiopurine Methyltransferase Activity in Postsurgical Patients With Crohn's Disease During 1 Year of Treatment With Azathioprine or Mesalazine , 2007, Therapeutic drug monitoring.

[19]  G. Schwarz.  Molybdenum cofactor biosynthesis and deficiency , 2005, Cellular and Molecular Life Sciences CMLS.

[20]  R. Weinshilboum,et al.  Thiopurine S-methyltransferase pharmacogenetics: variant allele functional and comparative genomics , 2005, Pharmacogenetics and genomics.

[21]  R. Weinshilboum,et al.  Human thiopurine S-methyltransferase pharmacogenetics: variant allozyme misfolding and aggresome formation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Michel Eichelbaum,et al.  Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants. , 2004, Pharmacogenetics.

[23]  J. Reiss,et al.  Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli. , 2004, Human molecular genetics.

[24]  V. Wray,et al.  The Tetrahydropyranopterin Structure of the Sulfur-free and Metal-free Molybdenum Cofactor Precursor* , 2004, Journal of Biological Chemistry.

[25]  Wee Hong Woo,et al.  Sulphite oxidase gene expression in human brain and in other human and rat tissues. , 2003, Biochemical and biophysical research communications.

[26]  J. Reiss,et al.  Molybdenum cofactor-deficient mice resemble the phenotype of human patients. , 2002, Human molecular genetics.

[27]  M. Eichelbaum,et al.  Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism. , 2002, Pharmacogenetics.

[28]  M Schwab,et al.  Comprehensive analysis of the genetic factors determining expression and function of hepatic CYP2D6. , 2001, Pharmacogenetics.

[29]  R. Mendel,et al.  Thiocarboxylation of Molybdopterin Synthase Provides Evidence for the Mechanism of Dithiolene Formation in Metal-binding Pterins* , 2001, The Journal of Biological Chemistry.

[30]  R. Mendel,et al.  Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[31]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[32]  M. Relling,et al.  Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. , 1999, Journal of the National Cancer Institute.

[33]  W. Evans,et al.  Enhanced proteasomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C. , 1999, Pharmacogenetics.

[34]  W. Evans,et al.  Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Hille.  The Mononuclear Molybdenum Enzymes. , 1997, Chemical reviews.

[36]  T. Garrow.  Purification, Kinetic Properties, and cDNA Cloning of Mammalian Betaine-Homocysteine Methyltransferase* , 1996, The Journal of Biological Chemistry.

[37]  R. Weinshilboum,et al.  Thiopurine methyltransferase: structure-activity relationships for benzoic acid inhibitors and thiophenol substrates. , 1986, Journal of medicinal chemistry.

[38]  K. Rajagopalan,et al.  The pterin component of the molybdenum cofactor. Structural characterization of two fluorescent derivatives. , 1984, The Journal of biological chemistry.

[39]  R. Weinshilboum,et al.  Human kidney thiopurine methyltransferase. Purification and biochemical properties. , 1983, Biochemical pharmacology.

[40]  K. Rajagopalan,et al.  Structural and metabolic relationship between the molybdenum cofactor and urothione. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A. Sakurai,et al.  Die Struktur des Urothions , 1969 .

[42]  U. John,et al.  Study of Health in Pomerania (SHIP): A health examination survey in an east German region: Objectives and design , 2005, Sozial- und Präventivmedizin.