Richard G.H. Cotton

Structural basis of autoregulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an α-β sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

THE BIOSYNTHESIS OF PHENYLALANINE AND TYROSINE; ENZYMES CONVERTING CHORISMIC ACID INTO PREPHENIC ACID AND THEIR RELATIONSHIPS TO PREPHENATE DEHYDRATASE AND PREPHENATE DEHYDROGENASE.

Abstract Cell-free extracts of Aerobacter aerogenes and Escherichia coli were chromatographed on DEAE-cellulose and the eluates examined for some of the enzymes concerned in the conversion of chorismic acid into phenylalanine and tyrosine. Enzymes which are able to convert chorismate into prephenate (chorismate mutase) are eluted in two peaks of activity. The effects of phenylalanine and tyrosine on enzyme levels and activity showed that one of these enzymes (chorismate mutase P) is probably related to phenylalanine biosynthesis and the other (chorismate mutase T) to tyrosine biosynthesis. Chorismate mutase T travelled on DEAE-cellulose with prephenate dehydrogenase, (prephenate; NAD oxidoreductase (decarboxylating) the subsequent enzymic activity on the tyrosine biosynthetic pathway. The two activities were also affected simultaneously by mutation. Chorismate mutase P travelled on columns with the next enzymic activity on the pathway of phenylalanine biosynthesis, prephenate dehydratase, (prephenate hydro-lyase (decarboxylating)) and both activities were affected following mutation. In extracts of the A. aerogenes strain examined, but not in E. coli extracts, a second peak of prephenate dehydratase activity was eluted in the early column fractions. This activity, unlike the prephenate dehydratase travelling with chorismate mutase P is not inhibited by phenylalanine. It is suggested that, in the biosynthesis of phenylalanine and tyrosine, there are two enzymes or enzyme complexes metabolising chorismate, one leading through prephenate to phenylpyruvate and the other leading through prephenate to 4-hydroxyphenylpyruvate.

Slowly but surely towards better scanning for mutations

3065, Aumum. As the number of dii genes described and the number of candi- date genes for diseases increases, so does the need for better DNA scan- ning methods. Notionally, the search for unknown mutations in DNA should be simple, thus, surely all one needs to do is to sequence the DNA. The reason why this does not appear to be true is complex, but perhaps the three major reasons are: (1) that the number of laboratories in the world who are expert sequencers is limited; (2) that many applications call for the analysis of hundreds of disease genes; and (3) that precise sequencing is time-consuming and expensive. These reasons have led to the development and refinement of a large number of DNA scanning methods in an attempt to develop economical and effective methods. There have been numerous articles reviewing and describing such meth- ods in recent years’-7. This work has been moving towards the elusive ‘ideal scanning method’ (Box 1). In the last year or so, there have been no dramatic novel approaches described, except perhaps the DHPLC (denaturing high-performance liquid chromatography) method, a hetero- duplex variant (see below), but there have been signiticant studies with, or advances made in, the methods described so far, and these advances are the subject of this article. An earlier issue of Trends

Variable phenotype of Alzheimer's disease with spastic paraparesis

A variant form of Alzheimers disease (AD), in which spastic paraparesis (SP) precedes dementia, is characterised by large, noncored, weakly neuritic Aβ‐amyloid plaques resembling cotton wool balls and is caused by genomic deletion of presenilin 1 exon 9. A pedigree with a 5.9 kb exon 9 deletion shows a phenotypic spectrum including subjects with typical AD or with SP and numerous cotton wool plaques. In SP subjects, dementia onset is delayed and modified. This phenotypic variation suggests that modifying factors are associated with exon 9 deletions. Ann Neurol 2001;49:125–129

Iminopeptiduria, skin ulcerations, and edema in a boy with prolidase deficiency

A 12-year-old boy with recurrent skin ulceration, chronic generalized lymphedema, and mild mental retardation was found to excrete massive amounts of dipeptides, most (but not all) of which had proline or hydroxyproline as the carboxyl terminal residue. Glycylproline predominated. Prolidase deficiency was demonstrated in red blood cells and in fibroblastic cells. Prolidase activity was present in continuous lymphoid cell cultures at the same low level observed in control cells.

Genetics: The human variome project

An ambitious plan to collect, curate, and make accessible information on genetic variations affecting human health is beginning to be realized.

Human Variome Project: an international collaboration to catalogue human genetic variation

1Division of Clinical Pharmacology, University of California, San Francisco, CA 94143, USA 2Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA 3Genomic Disorders Research Centre, 7th Floor Daly Wing, St. Vincent’s Hospital Melbourne, Australia 4University of Melbourne, Department of Medicine, St Vincents, Level 4, Clinical Sciences Building, Cnr Princes & Regent Streets, Fitzroy, Melbourne, Victoria 3065, Australia E-mail: cotton@unimelb.edu.au

The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection

Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.

General mutation databases: analysis and review

Databases of mutations causing Mendelian disease play a crucial role in research, diagnostic and genetic health care and can play a role in life and death decisions. These databases are thus heavily used, but only gene or locus specific databases have been previously reviewed for completeness, accuracy, currency and utility. We have performed a review of the various general mutation databases that derive their data from the published literature and locus specific databases. Only two—the Human Gene Mutation Database (HGMD) and Online Mendelian Inheritance in Man (OMIM)—had useful numbers of mutations. Comparison of a number of characteristics of these databases indicated substantial inconsistencies between the two databases that included absent genes and missing mutations. This situation strengthens the case for gene specific curation of mutations and the need for an overall plan for collection, curation, storage and release of mutation data.

Tetrahydrobiopterin, its mode of action on phenylalanine hydroxylase, and importance of genotypes for pharmacological therapy of phenylketonuria.

In about 20%–30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor tetrahydrobiopterin therapy. These patients can be diagnosed by an oral tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with tetrahydrobiopterin were categorized as predictive for tetrahydrobiopterin responsiveness or non‐responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.