Deletion of the Regulatory Domain in the Pyridoxal Phosphate-dependent Heme Protein Cystathionine β-Synthase Alleviates the Defect Observed in a Catalytic Site Mutant (1999)

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine β-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B6-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The ∼13-fold diminution in activity because of the mutation corresponds to an ∼7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine β-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B6-responsive phenotype associated with a cystathionine β-synthase-impaired homocystinuric genotype. The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine β-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B6-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The ∼13-fold diminution in activity because of the mutation corresponds to an ∼7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine β-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B6-responsive phenotype associated with a cystathionine β-synthase-impaired homocystinuric genotype. Elevated levels of homocysteine constitute an independent risk factor for two apparently unrelated pathologies: neural tube defects (1Mills J.L. McPartlin J.M. Kirke P.N. Lee Y.J. Conle M.R. Weir D.G. Lancet. 1995; 345: 149-151Abstract PubMed Scopus (500) Google Scholar) and cardiovascular diseases (2Refsum H. Ueland P.M. Nygard O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Crossref PubMed Scopus (1836) Google Scholar). Severe hyperhomocysteinemia results from autosomal recessive mutations in enzymes that metabolize intracellular homocysteine. Of these, the most common cause of homocystinuria is a functional deficiency in cystathionine β-synthase (3Mudd S.H. Levy H.L. Skovby F. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill, New York1995Google Scholar). This enzyme catalyzes the condensation of serine and homocysteine to give cystathionine. Its action commits homocysteine to degradation for ultimate removal as sulfate (4Finkelstein J.D. Martin J.J. J. Biol. Chem. 1984; 259: 9508-9513Abstract Full Text PDF PubMed Google Scholar). Cystathionine β-synthase is an unusual enzyme as it is dependent on two cofactors, heme and PLP, 1The abbreviations used are:PLPpyridoxal phosphateV168M-ΔC143truncated form of V168M cystathionine β-synthase lacking the C-terminal 143 residuesCBScystathionine β-synthaseCBSΔC143truncated cystathionine β-synthase with wild type sequenceAdoMetS-adenosylmethionine1The abbreviations used are:PLPpyridoxal phosphateV168M-ΔC143truncated form of V168M cystathionine β-synthase lacking the C-terminal 143 residuesCBScystathionine β-synthaseCBSΔC143truncated cystathionine β-synthase with wild type sequenceAdoMetS-adenosylmethionine for activity. In addition, the enzyme is regulated by the allosteric activator, AdoMet (5Finkelstein J.D. Kyle W.E. Martin J.J. Pick A.-M. Biochem. Biophys. Res. Commun. 1975; 66: 81-87Crossref PubMed Scopus (225) Google Scholar). The role of the PLP, in analogy with related enzymes that catalyze β-replacement reactions, is to anchor serine and activate it for thiolate addition following elimination of the hydroxyl group (6Borcsok E. Abeles R.H. Arch. Biochem. Biophys. 1982; 213: 695-707Crossref PubMed Scopus (54) Google Scholar). The binding site for PLP is in the N terminus extending from residues 68–209 and is readily identified by the significant homology to other PLP proteins (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). The PLP is bound as an internal aldimine to lysine 119 (8Kery V. Poneleit L. Meyer J.D. Manning M.C. Kraus J.P. Biochemistry. 1999; 38: 2710-2724Crossref Scopus (59) Google Scholar). The addition of serine to the enzyme results in fluorescence changes that are consistent with the formation of an unprotonated external aldimine and a second species that is tentatively identified as an aminoacrylate intermediate (9Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (96) Google Scholar). The role of the heme is less obvious. It is predicted to be bound to a sequence extending from residues 241–341 that is distantly related to other hemeproteins such as cytochrome P450 and sulfide dehydrogenase (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). Reduction of the ferric enzyme is correlated with a decrease in enzyme activity (10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), indicating that heme-linked oxidation state changes may regulate the activity of the enzyme. In addition, kinetic evidence indicates that the heme ligand, CO, binds competitively with respect to homocysteine, thus the binding of homocysteine must occur in the proximity of heme (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). This is also consistent with the small changes that are reported to occur in the heme Soret band upon addition of homocysteine (8Kery V. Poneleit L. Meyer J.D. Manning M.C. Kraus J.P. Biochemistry. 1999; 38: 2710-2724Crossref Scopus (59) Google Scholar). pyridoxal phosphate truncated form of V168M cystathionine β-synthase lacking the C-terminal 143 residues cystathionine β-synthase truncated cystathionine β-synthase with wild type sequence S-adenosylmethionine pyridoxal phosphate truncated form of V168M cystathionine β-synthase lacking the C-terminal 143 residues cystathionine β-synthase truncated cystathionine β-synthase with wild type sequence S-adenosylmethionine The native enzyme is a homotetramer and binds two hemes and four PLPs (9Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (96) Google Scholar, 10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). A truncated and highly active form of the enzyme, CBSΔC143 (lacking the C-terminal 143 amino acids), has been described recently (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar). It exists as a homodimer that binds two hemes and two PLPs but has lost its ability to bind and to be regulated by AdoMet (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). The binding site of AdoMet is predicted to lie in the so called “CBS domain” extending from residues 421–469. Both the full-length and truncated forms of cystathionine β-synthase bind half the equivalents of serine relative to PLP suggesting half of the sites activity (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar,9Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (96) Google Scholar). A number of mutations have been described in the cystathionine β-synthase gene from homocystinuric patients (12Kraus J.P. J. Inherit. Metab. Dis. 1994; 17: 383-390Crossref PubMed Scopus (85) Google Scholar). About 50% of these mutations result in a pyridoxine-responsive clinical phenotype,i.e. treatment with high doses of the PLP precursor, pyridoxine, is clinically effective (13Kraus J.P. Mato J.M. Caballero A. III Workshop on Methionine Metabolism. Consejo Superior de Investigaciones Cientificas, Madrid, Spain1996: 12-19Google Scholar). One such mutation is V168M (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar) in which a substitution in the predicted PLP binding domain (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar, 8Kery V. Poneleit L. Meyer J.D. Manning M.C. Kraus J.P. Biochemistry. 1999; 38: 2710-2724Crossref Scopus (59) Google Scholar) is detrimental to catalysis (Fig. 1). Remarkably, this mutation can be suppressed by a second one that causes premature termination at codon 409 and leads to the elimination of 143 amino acid residues from the C terminus (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar). The activity of this truncated mutant enzyme, designated hereafter as V168M-ΔC143 (Fig. 1), is sufficient to functionally complement the cystathionine β-synthase deficiency in yeast and is reported to be 13-fold higher than that of the full-length mutant, V168M (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar). The biochemical basis for the alleviation of the penalty imposed by a mutation in the catalytic domain, by deletion of the regulatory domain, is unknown. In this study, we have characterized the V168M and V168M-ΔC143 forms of cystathionine β-synthase and report on how interactions between the catalytic and regulatory domains dictate the PLP content of the enzyme. These studies provide the first biochemical explanation for pyridoxine responsiveness, a phenotype observed in a large number of homocystinuric patients. Bovine serum albumin, PLP, thrombin, 5,5′-dithiobis(nitrobenzene), l-homocysteine thiolactone, dithiothreitol, DEAE-Sepharose, and glutathione were obtained from Sigma. [14C]serine (158 mCi/mmol) was purchased from Amersham Pharmacia Biotech. GlutathioneS-transferase-Sepharose and Q-Sepharose (fast flow) were purchased from Amersham Pharmacia Biotech. Homocysteine was prepared from l-homocysteine thiolactone as reported previously (14Drummond J.T. Jarrett J. Gonzalez J.C. Huang S. Matthews R.G. Anal. Biochem. 1995; 228: 323-329Crossref PubMed Scopus (72) Google Scholar). AdoMet 1,4-butane disulfonate was a generous gift from Knoll Farmaceutici Spa (Milano, Italy). The full-length and truncated forms of the V168M mutant of human cystathionine β-synthase were purified as glutathioneS-transferase fusion proteins using recombinant expression systems (pGEXCBS1364 and pGEXCBS1364S, respectively) reported previously (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar). The constructs were a generous gift from Warren Kruger (Fox Chase Cancer Center, Philadelphia). The recombinantEscherichia coli were grown, and the enzymes were purified as described previously (10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) with the following exceptions. The cells were grown at 25 °C, and the initial pH of the Luria broth growth medium was adjusted to 8.5 with sodium hydroxide. The enzyme activity was assayed under aerobic conditions using 30 mm radiolabeled serine and 15 mm homocysteine as described previously (10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). For the V168M enzyme, severe substrate inhibition was observed beyond 10 mm homocysteine concentration. Therefore, only for the assays involving V168M, homocysteine was lowered from 15 mm, the concentration used in the standard assay, to 8 mm. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard. The concentration of stock homocysteine solution was determined immediately before the experiments by titration with dithiobis(nitrobenzene) and estimated using ε412 of 13,600 m−1cm−1. Steady-state kinetic analysis was performed in the aerobic assay as described previously (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). Unless noted otherwise, the reaction was initiated by the addition of 10 μg of enzyme rather than homocysteine. The reaction mixture was incubated at 37 °C for either 30 min or 1 h for assays containing V168M-CBSΔC143 and V168M, respectively. The reaction was stopped by the addition of 200 μl of 10% trifluoroacetic acid. The radiolabeled product,14C-labeled cystathionine, was separated from14C-labeled serine as described previously (10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). PLP was removed by incubating the enzyme (1.1 and 1.3 mg for V168M and V168M-CBSΔC143, respectively) in 100 mm potassium phosphate buffer, pH 7.2, containing 13 and 15 mm hydroxylamine (for V168M and V168M-CBSΔC143, respectively) for 3 days at 4 °C. The released oxime was separated from the protein by Centricon filtration, and the concentration of PLP in the filtrate was determined using a fluorimetric method described previously (9Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (96) Google Scholar). The metal content of CBS preparations was analyzed by plasma emission spectroscopy (15Jones J.B.J. Commun. Soil. Sci. Plant Anal. 1977; 8: 349-365Crossref Scopus (165) Google Scholar) at the Chemical Analysis Laboratory, University of Georgia, Athens. Twenty metal ions including the common transition metal ions are detectable by plasma emission spectroscopy. The full-length and truncated V168M mutants were purified using the procedures developed for the corresponding wild type enzymes (TableI). Significant differences were observed in the levels of expression of the two mutant proteins. The final yield from a 6-liter cell culture for V168M was ∼7 versus 23 mg for the full-length wild type enzyme. In contrast, the truncated protein carrying a mutation, V168M-ΔC143, was obtained in a significantly higher yield (220 mg) than the truncated wild type enzyme (70 mg) (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). The basis for the apparent differences in expression of the wild type and mutant enzymes is not The proteins used in these studies were The for the mutants reported in were determined using the standard assay 15 mm in which the enzyme is with serine (10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). these the wild type but not the mutant enzymes under for wild type and mutant cystathionine for the wild type enzyme were obtained from is activity as of cystathionine at 37 for the wild type enzyme were obtained from S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google is activity as of cystathionine at 37 °C. in a The full-length and truncated enzymes with the a homotetramer and the latter a homodimer (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). The cofactor is reported per in were to the PLP content of the full-length and truncated V168M mutant The V168M-ΔC143 enzyme binds two PLPs the truncated wild type enzyme, the full-length mutant, V168M, has PLPs versus 4 PLPs in wild type enzyme. The heme content of the two mutants was estimated from (Fig. and by plasma emission spectroscopy. Both mutant forms Soret at consistent with the of ferric The heme in the are from from the bound of the bound this analysis 23 for two hemes in the wild type a of is estimated for V168M, which is the as for the wild type enzyme The truncated enzymes with and the V168M mutation very similar (Fig. on an of (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar), a of is for V168M-ΔC143 The heme content of the mutant enzymes was by plasma emission which the of in V168M and in V168M-ΔC143, as has been observed for the corresponding proteins with wild type (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar, 10Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (224) Google of cofactor of wild type and mutant cystathionine were obtained from were obtained from These were obtained from S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google in a the V168M mutation was described in a B6-responsive homocystinuric the of PLP on the activity of the enzyme was examined The wild type enzyme is with PLP, so addition of this cofactor to the assay mixture has on enzyme activity as In contrast, the activity of V168M, which is with this upon the addition of The activity of V168M was under standard assay that the concentration of homocysteine was from 15 to 8 mm because of substrate the of substrate addition affects the activity of cystathionine β-synthase (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar), the assays used in these were initiated by the addition of enzyme rather than homocysteine. The activity of the truncated mutant V168M-ΔC143, is by the addition PLP, consistent with it with this cofactor The V168M mutant in to of PLP an estimated for PLP of of the of PLP and AdoMet to wild type and V168M cystathionine activity is reported as of for the wild type enzyme were under the assay conditions as for the mutants in which the reaction was initiated by the addition of enzyme rather than and activity is reported as of for the wild type enzyme were under the assay conditions as for the mutants in which the reaction was initiated by the addition of enzyme rather than homocysteine. in a The allosteric an in the activity of the V168M mutant that is similar to the with the wild type enzyme of enzyme activity by PLP and AdoMet is independent of are The truncated mutant, V168M-ΔC143, is by the of consistent with the of the allosteric binding domain upon elimination of the C-terminal (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). The concentration of the V168M and wild type enzyme on AdoMet were examined (Fig. is to be estimated significant differences were observed between the wild type and V168M mutant indicating that the mutation allosteric of cystathionine β-synthase. The kinetic of V168M was from that of the corresponding full-length wild type enzyme. severe substrate inhibition was observed at high homocysteine of the inhibition of enzyme activity 10 mm homocysteine, we were to the to an the for substrate inhibition in a reaction A. of Scholar). for serine at 8 for the full-length enzyme is 3 mm. In contrast, the kinetic properties of the truncated mutant, V168M-ΔC143, were similar not to reported for the corresponding wild type protein (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). of to in cystathionine β-synthase is correlated with homocystinuria (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar). This in the PLP binding domain (Fig. that is predicted on the high level of homology between cystathionine β-synthase and related PLP enzymes such as and (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). The mutation, V168M, has a on the PLP content the catalytic activity of cystathionine β-synthase. The PLP content is ∼7-fold than in the wild type enzyme. This decrease is in the 13-fold activity of V168M and with and respectively) versus the wild type and with and respectively) enzyme in the of PLP This be explained by half of the sites activity that is by cystathionine β-synthase (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar, S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (96) Google Scholar). The addition of PLP results in only partial restoration of enzyme activity. The of PLP of cystathionine β-synthase has been to be dependent on the of heme V. Kraus J.P. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). V168M has the complement of heme as the wild type enzyme, the of full restoration of enzyme activity in the of PLP be explained by heme In the V168M mutant may be to the level of activity as the wild type enzyme in the of PLP. under in the activity of V168M that is is than that of the wild type enzyme under similar conditions This activity be because of the severe substrate inhibition by homocysteine at beyond 10 mm, which is not with the wild type enzyme. The basis for this inhibition is unknown. the other we have been to wild type cystathionine β-synthase of PLP by addition of this cofactor (9Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (96) Google Scholar). the of activity may be a of cystathionine β-synthase that is common to both the wild type and the V168M enzyme The of the V168M mutation on PLP does not result from interactions between this and the cofactor binding it This is by the alleviation of the binding and associated catalytic imposed by the mutation by of the C-terminal regulatory The and enzyme thus V168M-ΔC143, is from the truncated wild type enzyme with The of the truncated wild type enzyme have been characterized in recently (7Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38 (in press)Crossref Scopus (9) Google Scholar). It two hemes and two PLPs as does V168M-ΔC143 and both and higher for the two removal of the C-terminal quarter of the protein that the regulatory AdoMet binding domain the of the V168M mutation on the full-length These to that is in interactions between the catalytic and regulatory domains and that this is important in wild type cystathionine β-synthase for the PLP and therefore activity The PLP domain of cystathionine β-synthase is to be very similar in to the corresponding domains in and are J. D. E. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). In from E. the corresponding is at in the is PLP such as and also have in this The containing in is not in with the PLP or the lysine in formation J. D. E. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and its therefore an on the binding of PLP, consistent with the in In these provide the first biochemical explanation for the B6-responsive phenotype associated with a homocystinuric genotype. The of the enzyme with PLP in V168M leads to a 13-fold level of enzyme activity. of PLP leads to a partial but not full of catalytic activity. These studies provide the interactions between the C-terminal regulatory and the catalytic domains that cofactor binding to the The detrimental of a number of mutations in cystathionine β-synthase have been reported to be by C-terminal (11Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (107) Google Scholar). The of these mutations to that may be in interactions between the regulatory and catalytic of the regulatory domain in the pyridoxal heme protein cystathionine β-synthase the observed in a catalytic site of line from the This recently described mutation is V168M in which a substitution in the predicted PLP binding domain is detrimental to catalysis (Fig. 1). It a phenotype in PDF