Purification, physicochemical and regulatory properties of serine hydroxymethyltransferase from sheep liver

Manohar, R.; Ramesh, K.; Rao, N. Appaji

doi:10.1007/bf02702579

Cited by 34 publications

(31 citation statements)

References 38 publications

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“…Enzyme Activity-SHMT activity of the purified recombinant proteins were determined as reported earlier (21,22 After incubation at 37°C for 20 min the reaction was stopped by the addition of 100 l of dimedone (400 mM in 50% ethanol). The reaction mixture was kept in boiling water bath for 5 min and the H-14 CHO-dimedone adduct was extracted into 3 ml of toluene.…”

Section: Methodsmentioning

confidence: 99%

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Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

Chaturvedi

Bhakuni

2003

Journal of Biological Chemistry

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From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with D-alanine resulting in formation of apoenzyme but L-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea-and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations.Recent years have seen increased incidence of tuberculosis in both developing and developed countries. Information available from the complete genome sequence of Mycobacterium tuberculosis has the potential of providing the information that would generate knowledge that will enable to elucidate the unusual biology of its etiological agent, M. tuberculosis.Serine hydroxymethyltransferase (SHMT), 1 L-serine:tetrahydrofolate 5,10-hydroxymethyltransferase is a pyridoxyl 5Ј-phosphate (PLP)-dependent enzyme. SHMT reaction plays a major role in cell physiology as it is considered to be a key enzyme in the pathway for interconversion of folate coenzymes that provide almost exclusively one-carbon fragments for the biosynthesis of a variety of end products such as DNA, RNA, ubiquinone, methionine,. The physiological role of SHMT is the reversible interconversion of serine to glycine and irreversible hydrolysis of 5,10-CH ϩ -H 4 PteGlu to 5-CHO-H 4 PteGlu. In addition to these physiological reactions, SHMT also catalyzes, in the absence of H 4 PteGlu, the retroaldol cleavage of several 3-hydroxyamino acids, such as allo-threonine, and the transamination and slow racemization of D-and Lalanine (4, 5).SHMT shows a ubiquitous distribution in nature, being found both in the prokaryotes and eukaryotes. In eukaryotes, SHMT exists as both cyt...

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Section: Methodsmentioning

confidence: 99%

“…1, A and B) and a single peak in ESI-MS (data not shown). The purified enzymes were assayed for SHMT activity (21,22) …”

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confidence: 99%

Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

Chaturvedi

Bhakuni

2003

Journal of Biological Chemistry

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“…The SHMT-catalysed aldol cleavage of serine in the presence of H % -folate was monitored with -[3-"%C]serine by the modified method standardized by Manohar et al [21]. The kinetic parameters for -serine and H % -folate and the aldolytic cleavage of -allothreonine were monitored as described by Jagath et al [22].…”

Section: Enzyme Assaysmentioning

confidence: 99%

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Talwar

Leelavathy

Rao

et al. 2000

Biochemical Journal

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Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

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“…Effect of L-cysteine on the activity of SHMT from mung bean seedlings, monkey (17), sheep (12), and human liver (24) is given in Table II. It is evident that the enzyme from all these sources was completely inhibited by L-cysteine (10 mM).…”

Section: Effect Of I-cysteine Treatment On the Enzyme Activitymentioning

confidence: 99%

“…Upon dialysis of the L-cysteine-treated enzyme, the activity was fully regained in the case of mung bean SHMT, and the addition of PLP (1 mM) did not enhance the activity of the enzyme. Although the activity of sheep (12), monkey (17), and human liver (24) SHMT were also inhibited by Lcysteine, the activity was not regained upon dialysis but was restored fully upon the addition of PLP (1 mM) (Table II).…”

Section: Effect Of I-cysteine Treatment On the Enzyme Activitymentioning

confidence: 99%

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5′-Phosphate-Dependent Enzyme

Sukanya

Vijaya²,

Savithri³

et al. 1991

Plant Physiol.

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Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5'-phosphate-containing enzyme, but the requirement of pyridoxal-5'-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of senne hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5'-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5'-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with L-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5'-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5'-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-D-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-Dseine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5'-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean seine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyndoxal-5'phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean seine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5'-phosphate that is present in the mammalian and bacterial enzymes.

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Purification, physicochemical and regulatory properties of serine hydroxymethyltransferase from sheep liver

Cited by 34 publications

References 38 publications

Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5′-Phosphate-Dependent Enzyme

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