Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 780131

Controlling protein evolution.

C Wills
View original source ↗Export metadata ↓

Abstract

A method is presented for selecting, with a high degree of specificity, mutants for the enzyme alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae having altered kinetics. The method depends on the facts that a) petite yeast strains (those unable to respire aerobically) have an absolute dependence on the presence of cytoplasmic alcohol dehydrogenase, and b) allyl alcohol is readily oxidized by yeast ADH to the highly poisonous product acrolein. If petite yeast are grown in the presence of allyl alcohol surviving mutants show alterations that can be traced to the ADH structural gene. Eight out of nine spontaneous mutant enzymes and five out of five mutants enzymes induced by nitrosoguanidine exhibited a slower electrophoresis mobility than wild type. There may therefore be some correlation between electrophoretic mobility and function. Of three mutant enzymes purified, one of spontaneous origin and two induced by nitrosoguanidine, each showed a different pattern of altered kinetics and a different specific activity. This strongly suggests that, despite the considerable specificity of the selective procedure, there are many ways in which an enzyme can be altered in response to an environmental stimulus. The implications of these results for the study of protein evolution and some directions for future research are briefly discussed.

Explore related subjects

Alcohol OxidoreductasesBiological EvolutionCytoplasmElectrophoresisKineticsMitochondriaMutationPolymorphism, GeneticProteinsSaccharomyces cerevisiaeJournal ArticleResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

C Wills. 1976. Controlling protein evolution.. https://pubmed.ncbi.nlm.nih.gov/780131/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1992

Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme.

6-Pyruvoyl-tetrahydropterin synthase (PTPS) is involved in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes such as the hepatic phenylalanine hydroxylase. BH4 deficiency causes malignant hyperphenylalaninemia. We cloned the human liver cDNA encoding PTPS. The coding region for PTPS contains 145 amino acids and predicts a polypeptide of 16'387 Da. The human amino acid sequence showed a 82% identity with the rat liver sequence. Expression of the cDNA in E. coli yielded the active enzyme and showed immunoreactivity with antibodies against the rat liver PTPS. This is the basis for the molecular understanding of BH4 deficiency in patients suffering from a defect in PTPS activity.

Alcohol Oxidoreductases↗
PubMed1992

The three-dimensional structures of methanol dehydrogenase from two methylotrophic bacteria at 2.6-A resolution.

The structures of methanol dehydrogenase (MEDH) from two closely related methylotrophic bacteria, Methylophilus methylotrophus and W3A1, have been determined at 2.6-A resolution. The molecule, a quinoprotein of molecular mass of about 138 kDa, contains two heavy (H) and two light (L) subunits of unknown sequence and two molecules of noncovalently associated pyrroloquinoline quinone. The two enzymes crystallize isomorphously in space group P2(1) with one H2L2 heterotetramer in the asymmetric unit. The electron density map of the M. methylophilus enzyme was obtained by multiple isomorphous replacement with anomalous scattering and improved by solvent leveling and electron density averaging. For model building, the amino acid sequence of MEDH from Paracoccus denitrificans for the H subunit and from Methylobacterium extorquens AM1 for the L subunit were used to represent the unknown amino acid sequence. At the present time, 579 and 57 amino acid residues for the large and small subunits, respectively, have been fitted into the map. The phases for MEDH from M. methylophilus were used directly to analyze the W3A1 structure, and both structures were refined to R-factors (where R = sigma[Fo-Fc[/sigma Fo) of 0.277 and 0.266, respectively. The L subunit contains a long alpha-helix and an extended N-terminal segment, both lying on the molecular surface of the H subunit. The H subunit contains eight antiparallel beta-sheets, each consisting of four strands arranged topologically like the letter W. The eight Ws are arranged circularly, forming the main disc-shaped body of the subunit, with some short helices and loops connecting the consecutive Ws, as well as some excursions within and between some of the Ws. The pyrroloquinoline quinone prosthetic group is located in the central channel of the large subunit near the surface of the molecule. The topology of the eight-W folding unit is similar to those of the six- and seven-W folding units previously reported for three other proteins, neuraminidase, methylamine dehydrogenase, and galactose oxidase.

Alcohol Oxidoreductases↗
PubMed1992

Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion.

Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer. The role of Ca2+ was investigated using the MDH from Methylobacterium extorquens. This was shown to be similar to the MDH from Hyphomicrobium X in having 2 mol of prosthetic group (pyrroloquinoline quinine; PQQ) per mol of tetramer, the PQQ being predominantly in the semiquinone form. MDH isolated from the methanol oxidation mutants MoxA-, K- and L- contained no Ca2+. They were identical with the enzyme isolated from wild-type bacteria with respect to molecular size, subunit configuration, pI, N-terminal amino acid sequence and stability under denaturing conditions (low pH, high urea and high guanidinium chloride) and in the nature and content of the prosthetic group (2 mol of PQQ per mol of MDH). They differed in their lack of Ca2+, the oxidation state of the extracted PQQ (fully oxidized), absence of the semiquinone form of PQQ in the enzyme, reactivity with the suicide inhibitor cyclopropanol and absorption spectrum, which indicated that PQQ is bound differently from that in normal MDH. Incubation of MDH from the mutants in calcium salts led to irreversible time-dependent reconstitution of full activity concomitant with restoration of a spectrum corresponding to that of fully reduced normal MDH. It is concluded that Ca2+ in MDH is directly or indirectly involved in binding PQQ in the active site. The MoxA, K and L proteins may be involved in maintaining a high Ca2+ concentration in the periplasm. It is more likely, however, that they fill a 'chaperone' function, stabilizing a configuration of MDH which permits incorporation of low concentrations of Ca2+ into the protein.

Alcohol Oxidoreductases↗