Information

Biochemical and Genetic Identifcation of Enzymes - Biology

Biochemical and Genetic Identifcation of Enzymes - Biology



We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Biochemical and Genetic Identifcation of Enzymes

World's First Artificial Enzymes Created From Synthetic Genetic Material

Scientists have made a breakthrough in the field of synthetic biology by creating, for the first time, enzymes from artificial genetic material that does not exist in nature. This exciting new work not only offers new insights into the origins of life on Earth, but also has implications for our search for extraterrestrial life on other planets.

The foundationsਏor this study were laid a couple of years ago when UK scientists created synthetic versions of DNA, the molecule that carries the genetic information of all living things on Earth, and its close chemical cousin, RNA. This synthetic genetic material was created using the same building blocks that are found in DNA and RNA, but the scientists strung them together with different molecules. These resulting synthetic molecules, which were dubbed ‘XNAs,’ or xeno nucleic acid, were found to be capable of storing and passing on genetic information.

Although it was widely believed that DNA and RNA, together with proteins, were the only molecules that could form enzymes, the same researchers have now demonstrated that it is possible to create synthetic enzymes using only these XNAs. These molecules, which have been named ‘XNAzymes,’ were capable of chopping up and stitching together bits of RNA, just like natural enzymes. One of them was even capable of joining up fragments of XNA.

Enzymes, nature’s catalysts, are fundamental to life on Earth because almost all of the biochemical reactions taking place in cells are inefficient at ambient temperatures. Enzymes are therefore required to give reactions, such as synthesizing DNA or digesting food, a kick-start, allowing them to occur at rates sufficient for life to exist.

Although the majority of enzymes are proteins, some RNA molecules possess catalytic activity. It’s widely believed that the evolution of early pieces of genetic information, which may have been RNA, into self-replicating enzymes was likely a key event in the emergence of life on Earth. This work is therefore important because it recreates one of the earliest stages towards life. However, it also teases us with the possibility that life could evolve without DNA or RNA, which are widely regarded as the prerequisites for life.

“Our work suggests that, in principle, there are a number of possible alternatives to nature’s molecules that will support the catalytic processes required for life,” said lead scientist Philip Holliger. “Life’s 𠆌hoice’ of RNA and DNA may just be an accident of prehistoric chemistry.”

Because it is possible to create genetic material and enzymes from building blocks that don’t occur naturally, this suggests that life could emerge from different molecular backbones on other planets. This could therefore “potentially widen the number of exoplanets that one could consider would be hospitable for some form of life,” according to Holliger.

This work, the researchers say, could also lead to a new wave of treatments for a variety of diseases.ਊs explained by Dr. Holliger, it may be possible to synthesize XNAs that are capable of chopping up pieces of RNA produced from cancer genes or fragments of viral RNA. And because the XNAs don’t occur naturally, it is unlikely that they will be recognized and destroyed by other enzymes in the body.


Relationship between Gene and Enzyme | Cell Biology

An understanding of the relationship of the gene and the enzyme is crucial for an analysis of the basis of growth and development of an orga­nism. It has been established through varied lines of evidences that genes control all hereditary characters of an organism, which are transmitted from generation to generation.

Simultaneously, it is also established that all essential biochemical pathways, responsible for the expression of the character, are controlled by the enzymes. It has been demonstrated that genetic control of all characters is mediated through specific enzymes. More precisely, the message of gene is ultimately carried out by the enzymes.

Just as every biochemical pathway is con­trolled by a gene, enzyme too responsible for every step in metabolism. Just as genes are spe­cific for each and every character, enzymes too are specific for each step in metabolism. The dif­ference between different genes is also reflected in the basic chemical differences of the enzyme which they control.

One Gene-One Enzyme Hypothesis:

A wild strain of Neurospora crassa can synthesize all the amino acids essential for the formation of its protein.

Normally, this mould can grow in a ‘minimal medium’ containing sucrose, nitrate, minerals and only vitamin – biotin. However, if spores of wild strain are treated with a mutagen, some of the spores will be unable to grow on the minimal medium and will require the addition of some other substance, such as amino acid citrulline or vitamin niacin.

In a living system, the synthesis of organic molecules, such as amino acids, is termed biosyn­thesis. It entails in most cases a series of stepwise biochemical reactions, each catalyzed by an enzyme. The sequential reactions that lead to the synthesis of a given compound, such as arginine – constitute a biosynthetic pathway.

The cycle from ornithine to arginine can be summarized as follows:

Beadle and Tatum while studying on several mutant strains of Neurospora found that they were unable to synthesize arginine. Since there are different steps in the synthesis of arginine, a mutation at any one of them results in a block in arginine biosynthesis.

When each mutant type is crossed with a normal or wild type, the later progeny shows the inheritance according to a monohybrid ratio, indicating a single gene diffe­rence.

One class of mutant was found to grow on arginine or citrulline and also on ornithine. An arginine-requiring mutant that can grow in the presence of ornithine is able to carry out the reactions from ornithine through citrulline to arginine.

Therefore, a mutant of this type will also grow when given either citrulline or argi­nine. Obviously, gene mutation in this class of mutant results in a block in one of the reactions that precedes the synthesis of ornithine.

Similarly, a mutant that can grow in the presence of citru­lline or arginine but not in ornithine, must be unable to convert ornithine into cirtrulline and accordingly a mutant that can utilize only argi­nine must possess a block between citrulline and arginine. The evidence derived from examining the biosynthesis shows that each of the three mutant classes has lost one of the three different biochemical reactions.

A different enzyme is required for each of the steps in the biosynthesis of arginine. Therefore, the functional distinction between wild and mutant types, resides either in the loss of ability to produce one of the three enzymes essential for arginine biosynthesis or in the production of an altered enzyme. In this case, therefore, the relation between gene and enzyme is 1:1 (Fig. 14.1).

Inborn Metabolic Errors in Human:

One of the earliest investigations of biochemical muta­tions concerned with the metabolic disease of human known as alkaptonuria where the urine is black. This metabolic error is inherited as a recessive trait. The affected individuals are unable to metabolize a substance called homogentisic acid into acetoacetic acid, which in turn is excreted in the urine.

A single bio­chemical reaction is missing in alkaptonurics, for which they cannot convert homogentisic acid into acetoacetic acid.

Homogentisic acid is as such excreted in the urine and oxidized upon exposure to air causing the urine to become black. The chemical reaction by which homo­gentisic acid is converted to acetoacetic acid requires the presence of an enzyme that is either inactive or absent in alkaptonurics (Fig. 14.2).

One Gene-One Polypeptide Con­cept:

The ‘one gene-one enzyme’ hypothesis has been modified in several different ways. The gene forms a mRNA molecule which serves for coding of protein (enzyme). In some cases seve­ral genes form a single mRNA strand, which is then said to be polycistronic. As a single mRNA may lead to the production of several poly­peptides from a single polycistronic structure, the recognized concept is one gene-one polypep­tide.

This has been illustrated by the study of sick­le cell anaemia in human beings, found specially in Negroes. The R.B.C. in this disease become sickle shaped owing to the lower concentration of oxygen. This causes rupture of cells and severe haemolytic anaemia. The molecular basis to this lies in the difference of arrangement of amino acid molecules in haemoglobin.

Haemoglobin is a protein, consists of four polypeptide chains – two identical a-chains and two identical p-chains. The sickle shaped haemoglobin (HbS) differs from normal haemoglobin (HbA) in the presence of valine in the place of glutamic acid at sixth position of one of the p-chain (Fig. 14.3).

Sickle cell anaemia is, therefore, produced by a single change in any one of the two β- polypeptide chains, caused by a single mutation in one gene, it means that one gene controls the synthesis of one polypeptide chain and not com­plete protein.


Protein Biochemistry in the Early 1940s

It is so obvious to us today that proteins are linear polymers of amino acids arranged in a specific sequence and connected by peptide bonds that it is difficult to put oneself in the mind-set of a student of biochemistry in the years around 1940. To do so, it is instructive to look at successive editions of standard textbooks written in this period (Bodansky 1938 Harrow 1935, 1940, 1943, 1947, 1950, 1954, 1958, 1962, 1966). Not only were there some odd notions, to our eyes, about the nature of protein structure, but also many assumed that the genes themselves were proteins. The second (1940) edition of the text by Harrow discusses the structure of proteins in terms of the Bergmann–Niemann hypothesis of repeating 288 amino acid units. They supposed that the proteins were constructed along simple lines with a limited number of amino acids repeating at regular intervals. Also seriously considered was the “cyclol” hypothesis of Dorothy Wrinch, which supposed the proteins to be made up of a series of hexagonal structures composed of amino acids. Stanley’s crystallization of tobacco mosaic virus (TMV) was discussed as an example of the genetic role of protein. The Bergmann hypothesis was still discussed in the third (1943) edition of Harrow although the cyclol hypothesis had been discarded. TMV was recognized as a nucleoprotein but not much was made of the nucleo portion. The fourth edition (1947) did not change much. The fifth (1950) edition no longer discusses either the Bergmann or the cyclol hypothesis.

The importance of hydrogen bonds in determining the three-dimensional structure of molecules, in particular proteins, had been recognized, however. In 1942, Linus Pauling published an article in the Journal of Experimental Medicine claiming the formation of specific antibodies by denaturing and renaturing globulin around an antigen (Pauling and Campbell 1942). As late as 1954, Chemical Reviews could publish an article on the “Microheterogeneity of Proteins” (Colvin et al. 1954).

Why was the connection between metabolic reactions and genetic factors not given more attention? One explanation might be that it was unclear what to do about it. That is, it was not obvious how the problem could be approached experimentally. After all, alkaptonuria and the other conditions discussed in Archibald Garrod’s book Inborn Errors of Metabolism (Garrod 1909) were rare and, as he stressed, not life threatening. It was not clear how these observations on a metabolic oddity fit into a general pattern linking biochemistry and genetics, especially since it was uncertain whether the “gene” was a molecule or an organelle and the basis of enzyme specificity could not even be guessed, given the then current knowledge of protein structure.


Abstract

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.


Useful Links

This is one of over 2,400 courses on OCW. Explore materials for this course in the pages linked along the left.

MIT OpenCourseWare is a free & open publication of material from thousands of MIT courses, covering the entire MIT curriculum.

No enrollment or registration. Freely browse and use OCW materials at your own pace. There's no signup, and no start or end dates.

Knowledge is your reward. Use OCW to guide your own life-long learning, or to teach others. We don't offer credit or certification for using OCW.

Made for sharing. Download files for later. Send to friends and colleagues. Modify, remix, and reuse (just remember to cite OCW as the source.)

About MIT OpenCourseWare

MIT OpenCourseWare is an online publication of materials from over 2,500 MIT courses, freely sharing knowledge with learners and educators around the world. Learn more »

© 2001&ndash2018
Massachusetts Institute of Technology

Your use of the MIT OpenCourseWare site and materials is subject to our Creative Commons License and other terms of use.


Biochemical and Genetic Identifcation of Enzymes - Biology

Methane fermentation is a versatile biotechnology capable of converting almost all types of polymeric materials to methane and carbon dioxide under anaerobic conditions. This is achieved as a result of the consecutive biochemical breakdown of polymers to methane and carbon dioxide in an environment in which a variety of microorganisms which include fermentative microbes (acidogens) hydrogen-producing, acetate-forming microbes (acetogens) and methane-producing microbes (methanogens) harmoniously grow and produce reduced end-products. Anaerobes play important roles in establishing a stable environment at various stages of methane fermentation.

Methane fermentation offers an effective means of pollution reduction, superior to that achieved via conventional aerobic processes. Although practiced for decades, interest in anaerobic fermentation has only recently focused on its use in the economic recovery of fuel gas from industrial and agricultural surpluses.

The biochemistry and microbiology of the anaerobic breakdown of polymeric materials to methane and the roles of the various microorganisms involved, are discussed here. Recent progress in the molecular biology of methanogens is reviewed, new digesters are described and improvements in the operation of various types of bioreactors are also discussed.

Methane fermentation is the consequence of a series of metabolic interactions among various groups of microorganisms. A description of microorganisms involved in methane fermentation, based on an analysis of bacteria isolated from sewage sludge digesters and from the rumen of some animals, is summarized in Fig. 4-1. The first group of microorganisms secrete enzymes which hydrolyze polymeric materials to monomers such as glucose and amino acids, which are subsequently converted to higher volatile fatty acids, H 2 and acetic acid (Fig. 4-1 stage 1). In the second stage, hydrogen-producing acetogenic bacteria convert the higher volatile fatty acids e.g., propionic and butyric acids, produced, to H 2 , CO 2 , and acetic acid. Finally, the third group, methanogenic bacteria convert H 2 , CO 2 , and acetate, to CH 4 and CO 2 .

Polymeric materials such as lipids, proteins, and carbohydrates are primarily hydrolyzed by extracellular, hydrolases, excreted by microbes present in Stage 1 (Fig. 4-1). Hydrolytic enzymes, (lipases, proteases, cellulases, amylases, etc.) hydrolyze their respective polymers into smaller molecules, primarily monomeric units, which are then consumed by microbes. In methane fermentation of waste waters containing high concentrations of organic polymers, the hydrolytic activity relevant to each polymer is of paramount significance, in that polymer hydrolysis may become a rate-limiting step for the production of simpler bacterial substrates to be used in subsequent degradation steps.

Lipases convert lipids to long-chain fatty acids. A population density of 10 4 - 10 5 lipolytic bacteria per ml of digester fluid has been reported. Clostridia and the micrococci appear to be responsible for most of the extracellular lipase producers. The long-chain fatty acids produced are further degraded by p-oxidation to produce acetyl CoA.

Proteins are generally hydrolyzed to amino acids by proteases, secreted by Bacteroides, Butyrivibrio, Clostridium, Fusobacterium, Selenomonas, and Streptococcus. The amino acids produced are then degraded to fatty acids such as acetate, propionate, and butyrate, and to ammonia as found in Clostridium, Peptococcus, Selenomonas, Campylobacter, and Bacteroides.

Polysaccharides such as cellulose, starch, and pectin are hydrolyzed by cellulases, amylases, and pectinases. The majority of microbial cellulases are composed of three species: (a) endo-(3-l,4-glucanases (b) exo-p-l,4-glucanases (c) cellobiase or p-glucosidase. These three enzymes act synergistically on cellulose effectively hydrolyzing its crystal structure, to produce glucose. Microbial hydrolysis of raw starch to glucose requires amylolytic activity, which consist of 5 amylase species: (a) a-amylases that endocleave a ±1-4 bonds (b) p-amylases that exocleave a ±1-4 bonds (c) amyloglucosidases that exocleave a ±l-4 and a ±l-6 bonds (d) debranching enzymes that act on a ±l-6 bonds (e) maltase that acts on maltose liberating glucose. Pectins are degraded by pectinases, including pectinesterases and depolymerases. Xylans are degraded with a ²-endo-xylanase and a ²-xylosidase to produce xylose.

Hexoses and pentoses are generally converted to C 2 and C 3 intermediates and to reduced electron carriers (e.g., NADH) via common pathways. Most anaerobic bacteria undergo hexose metabolism via the Emden-Meyerhof-Parnas pathway (EMP) which produces pyruvate as an intermediate along with NADH. The pyruvate and NADH thus generated, are transformed into fermentation endo-products such as lactate, propionate, acetate, and ethanol by other enzymatic activities which vary tremendously with microbial species.

Thus, in hydrolysis and acidogenesis (Fig. 4-1 Stage 1), sugars, amino acids, and fatty acids produced by microbial degradation of biopolymers are successively metabolised by fermentation endo-products such as lactate, propionate, acetate, and ethanol by other enzymatic activities which vary tremendously with microbial species.

Thus, in hydrolysis and acidogenesis (Fig. 4-1 Stage 1), sugars, ammo acids, and fatty acids produced by microbial degradation of biopolymers are successively metabolised by groups of bacteria and are primarily fermented to acetate, propionate, butyrate, lactate, ethanol, carbon dioxide, and hydrogen (2).

Although some acetate (20%) and H 2 (4%) are directly produced by acidogenic fermentation of sugars, and amino acids, both products are primarily derived from the acetogenesis and dehydrogenation of higher volatile fatty acids (Fig. 4-1 Stage 2).

Obligate H 2 -producing acetogenic bacteria are capable of producing acetate and H 2 from higher fatty acids. Only Syntrophobacter wolinii, a propionate decomposer (3) and Sytrophomonos wolfei, a butyrate decomposer (4) have thus far been isolated due to technical difficulties involved in the isolation of pure strains, since H 2 produced, severely inhibits the growth of these strains. The use of co-culture techniques incorporating H 2 consumers such as methanogens and sulfate-reducing bacteria may therefore facilitate elucidation of the biochemical breakdown of fatty acids.

Overall breakdown reactions for long-chain fatty acids are presented in Tables 4-1 and 4-2. H 2 production by acetogens is generally energetically unfavorable due to high free energy requirements ( a ”G o, > 0 Table 4-1 and 4-2). However, with a combination of H 2 -consuming bacteria (Table 4-2, 4-3), co-culture systems provide favorable conditions for the decomposition of fatty acids to acetate and CH 4 or H 2 S ( a ”G o, < 0). In addition to the decomposition of long-chain fatty acids, ethanol and lactate are also converted to acetate and H 2 by an acetogen and Clostridium formicoaceticum, respectively.

The effect of the partial pressure of H 2 on the free energy associated with the conversion of ethanol, propionate, acetate, and H 2 /CO 2 during methane fermentation is shown in Fig. 4-2. An extremely low partial pressure of H 2 (10 -5 atm) appears to be a significant factor in propionate degradation to CH 4 . Such a low partial pressure can be achieved in a co-culture with H 2 -consuming bacteria as previously described (Table 4-2,4-3).

Methanogens are physiologically united as methane producers in anaerobic digestion (Fig. 4-1 Stage 3). Although acetate and H 2 /CO 2 are the main substrates available in the natural environment, formate, methanol, methylamines, and CO are also converted to CH 4 (Table 4-3).

Table 4-1 Proposed Reactions Involved in Fatty Acid Catabolism by Syntrophomonas wolfei

+ 2 H 2 O 2 CH 3 COO - + 2H 2 + H +

CH 3 CH 2 CH 2 CH 2 CH 2 COO -

+ 4 H 2 O 3 CH 3 COO - + 4H 2 + 2H +

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COO -

+ 6 H 2 O 4 CH 3 COO - + 6H 2 + 3H +

+1 H 2 O CH 3 CH 2 COO - + CH 3 COO - +2 H 2 + H +

CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 COO -

+ 4 H 2 O CH 3 CH 2 COO - + 2 CH 3 COO - +4 H 2 + 2H +

CH 3 CHCH 2 CH 2 CH 2 COO -
|
CH 3

+ 2 H 2 O CH 3 CHCH 2 COO - + CH 3 COO - + 2H 2 + H +
|
CH 3

Table 4-2 Free-Energy Changes for Reactions Involving Anaerobic Oxidation in Pure Cultures or in Co-Cultures with H 2 -Utilizing Methanogens or Desulfovibrio spp.

1. Proton-reducing (H 2 -producing) acetogenic bacteria

A. CH 3 CH 2 CH 2 COO - + 2H 2 O 2 CH 3 COO - + 2H 2 + H +

B. CH 3 CH 2 COO - + 3H 2 O CH 3 COO - + HCO 3 - + H + + 3H 2

2. H 2 -using methanogens and desulfovibrios

C. 4H 2 + HCO 3 - + H + CH 4 + 3 H 2 O

D. 4H 2 + S0 4 2- + H + HS - + 4 H 2 O

A + C 2 CH 3 CH 2 CH 2 COO - + HCO 3 - + H 2 O 4 CH 3 COO - + H + + CH 4

A + D 2 CH 3 CH 2 CH 2 COO - + S0 4 2- 4 CH 3 COO - + H + + HS -

B + C 4 CH 3 CH 2 COO - + 12H 2 4 CH 3 COO - + HCO 3 - + H + + 3 CH 4

B + D 4 CH 3 CH 2 COO - + 3 S0 4 2 " 4 CH 3 COO - + 4 HCO 3 - + H + + 3 HS -

Table 4-3 Energy-Yielding Reactions of Methanogens

CO 2 + 4 H 2 ® CH 4 + 2H 2 O

HCO 3 - + 4 H 2 + H + ® CH 4 + 3 H 2 O

CH 3 COO - + H 2 O ® CH 4 + HCO 3 -

HCOO - + H + ® 0.25 CH 4 + 0.75 CO 2 + 0.5 H 2 O

CO + 0.5 H 2 O ® 0.25 CH 4 + 0.75 CO 2

CH 3 OH ® 0.75 CH 4 + 0.25 CO 2 + 0.5 H 2 O

CH 3 NH 3 + + 0.5 H 2 O ® 0.75 CH 4 + 0.25 CO 2 + NH 4 +

(CH 3 ) 2 NH 2 + + H 2 O ® 1.5 CH 4 + 0.5 CO 2 + NH 4 +

(CH 3 ) 2 NCH 2 CH 3 H + + H 2 O ® 1.5 CH 4 + 0.5 CO 2 + + H 3 NCH 2 CH 3

(CH 3 ) 3 NH+ 1.5H 2 O ® 2.25 CH 4 + 0.75 CO 2 + NH 4 +

Since methanogens, as obligate anaerobes, require a redox potential of less than -300 mV for growth, their isolation and cultivation was somewhat elusive due to technical difficulties encountered in handling them under completely O 2 -free conditions. However, as a result of a greatly improved methanogen isolation techniques developed by Hungate (6), more than 40 strains of pure methanogens have now been isolated. Methanogens can be divided into two groups: H 2 /CO 2 - and acetate-consumers. Although some of the H 2 /CO 2 -consumers are capable of utilizing formate, acetate is consumed by a limited number of strains, such as Methanosarcina spp. and Methanothrix spp. (now, Methanosaeta), which are incapable of using formate. Since a large quantity of acetate is produced in the natural environment (Fig. 4-1), Methanosarcina and Methanothrix play an important role in completion of anaerobic digestion and in accumulating H 2 , which inhibits acetogens and methanogens. H 2 -consuming methanogens are also important in maintaining low levels of atmospheric H 2 .

H 2 /CO 2 -consuming methanogens reduce CO 2 as an electron acceptor via the formyl, methenyl, and methyl levels through association with unusual coenzymes, to finally produce CH 4 (7) (Fig. 4-3). The overall acetoclastic reaction can be expressed as:

Since a small part of the CO 2 is also formed from carbon derived from the methyl group, it is suspected that the reduced potential produced from the methyl group may reduce CO 2 to CH 4 (8).

On the basis of homologous sequence analysis of 16S rRNAs, methanogens have been classified into one of the three primary kingdoms of living organisms: the Archaea (Archaebacteria). The Archaea also include major groups of organisms such as thermophiles and halophiles. Although Archaea possess a prokaryotic cell structure and organization, they share common feature with eukaryotes: homologous sequences in rRNA and tRNA, the presence of inn-ones in their genomes, similar RNA polymerase subunit organization, immunological homologies, and translation systems.

Recombinant DNA technology is one of the most powerful techniques for characterizing the biochemical and genetic regulation of methanogenesis. This necessitates the selection of genetic markers, an efficient genetic transformation system, and a vector system for genetic recombination as prerequisites.

Genetically marked strains are prerequisites for genetic studies: these strains can be employed to develop a genetic-exchange system in methanogens based on an efficient selection system. Since growth of M. thermoautotrophicum is inhibited by fluorouracil, analogue-resistant strains were isolated by spontaneous mutation. Other mutants resistant to DL-ethionine or 2-bromoethane sulfonate (coenzyme M analogue), in addition to autotrophic mutants, were obtained by mutagenic treatment. Several autotrophic strains were also obtained for the acetoclastic methanogen, M. voltae. These mutant strains are listed in Table 4-4.

Although some methanogen genes such as amino acid and purine biosysnthetic genes, transcription and translation machinery genes, and structural protein genes, have been cloned, genes encoding enzymes involved in methanogenesis were chosen as "methane genes" here.

Methyl CoM reductase (MR Fig. 4-3) constitutes approximately 10% of the total protein in methanogenic cultures. The importance and abundance of MR inevitably focused initial attention on elucidating its structure and the mechanisms directing its synthesis and regulation. MR- encoding genes have been cloned and sequenced from Methanococcus vanielli, M. voltae, Methanosarcina barkeri, Methanobacterium thermoautotrophicum and M. fervidus.

Formylmethanofuran transferase (FTR) catalyzes the transfer of a formyl group from formylmethanofuran (MFR) to tetrahydromethanopterin (H 4 MPT) (Fig. 4-3, 4-2). The FTR-encoding gene from M. thermoautotrophicum has been cloned, sequenced, and functionally expressed in E. coli. Formate dehydrogenase (FDH) may sometimes account for 2 to 3% of the total soluble proteins in methanogenic cultures. The two genes encoding the a ± and a ² subunits of FDH have been cloned and sequenced from M formicicum. In addition, the genes encoding F 420 -reducing hydrogenase (Fig. 4-3), ferredoxin, and ATPase have also been cloned.

Table 4-4 Auxotrophic and Drug-Resistant Mutants Applicable To Gene Transfer Experiments


Contents

Although some instances of errors in metabolism following Mendelian inheritance patterns were known earlier, beginning with the 1902 identification by Archibald Garrod of alkaptonuria as a Mendelian recessive trait, for the most part genetics could not be applied to metabolism through the late 1930s. Another of the exceptions was the work of Boris Ephrussi and George Beadle, two geneticists working on the eye color pigments of Drosophila melanogaster fruit flies in the Caltech laboratory of Thomas Hunt Morgan. In the mid-1930s they found that genes affecting eye color appeared to be serially dependent, and that the normal red eyes of Drosophila were the result of pigments that went through a series of transformations different eye color gene mutations disrupted the transformations at a different points in the series. Thus, Beadle reasoned that each gene was responsible for an enzyme acting in the metabolic pathway of pigment synthesis. However, because it was a relatively superficial pathway rather than one shared widely by diverse organisms, little was known about the biochemical details of fruit fly eye pigment metabolism. Studying that pathway in more detail required isolating pigments from the eyes of flies, an extremely tedious process. [6]

After moving to Stanford University in 1937, Beadle began working with biochemist Edward Tatum to isolate the fly eye pigments. After some success with this approach—they identified one of the intermediate pigments shortly after another researcher, Adolf Butenandt, beat them to the discovery—Beadle and Tatum switched their focus to an organism that made genetic studies of biochemical traits much easier: the bread mold Neurospora crassa, which had recently been subjected to genetic research by one of Thomas Hunt Morgan's researchers, Carl C. Lingegren. Neurospora had several advantages: it required a simple growth medium, it grew quickly, and because of the production of ascospores during reproduction it was easy to isolate genetic mutants for analysis. They produced mutations by exposing the fungus to X-rays, and then identified strains that had metabolic defects by varying the growth medium. This work of Beadle and Tatum led almost at once to an important generalization. This was that most mutants unable to grow on minimal medium but able to grow on “complete” medium each require addition of only one particular supplement for growth on minimal medium. If the synthesis of a particular nutrient (such as an amino acid or vitamin) was disrupted by mutation, that mutant strain could be grown by adding the necessary nutrient to the medium. This finding suggested that most mutations affected only a single metabolic pathway. Further evidence obtained soon after the initial findings tended to show that generally only a single step in the pathway is blocked. Following their first report of three such auxotroph mutants in 1941, Beadle and Tatum used this method to create series of related mutants and determined the order in which amino acids and some other metabolites were synthesized in several metabolic pathways. [7] The obvious inference from these experiments was that each gene mutation affects the activity of a single enzyme. This led directly to the one gene–one enzyme hypothesis, which, with certain qualifications and refinements, has remained essentially valid to the present day. As recalled by Horowitz et al., [8] the work of Beadle and Tatum also demonstrated that genes have an essential role in biosyntheses. At the time of the experiments (1941), non-geneticists still generally believed that genes governed only trivial biological traits, such as eye color, and bristle arrangement in fruit flies, while basic biochemistry was determined in the cytoplasm by unknown processes. Also, many respected geneticists thought that gene action was far too complicated to be resolved by any simple experiment. Thus Beadle and Tatum brought about a fundamental revolution in our understanding of genetics.

The nutritional mutants of Neurospora also proved to have practical applications in one of the early, if indirect, examples of military funding of science in the biological sciences, Beadle garnered additional research funding (from the Rockefeller Foundation and an association of manufacturers of military rations) to develop strains that could be used to assay the nutrient content of foodstuffs, to ensure adequate nutrition for troops in World War II. [9]

In their first Neurospora paper, published in the November 15, 1941, edition of the Proceedings of the National Academy of Sciences, Beadle and Tatum noted that it was "entirely tenable to suppose that these genes which are themselves a part of the system, control or regulate specific reactions in the system either by acting directly as enzymes or by determining the specificities of enzymes", an idea that had been suggested, though with limited experimental support, as early as 1917 they offered new evidence to support that view, and outlined a research program that would enable it to be explored more fully. [1] By 1945, Beadle, Tatum and others, working with Neurospora and other model organisms such as E. coli, had produced considerable experimental evidence that each step in a metabolic pathway is controlled by a single gene. In a 1945 review, Beadle suggested that "the gene can be visualized as directing the final configuration of a protein molecule and thus determining its specificity." He also argued that "for reasons of economy in the evolutionary process, one might expect that with few exceptions the final specificity of a particular enzyme would be imposed by only one gene." At the time, genes were widely thought to consist of proteins or nucleoproteins (although the Avery–MacLeod–McCarty experiment and related work was beginning to cast doubt on that idea). However, the proposed connection between a single gene and a single protein enzyme outlived the protein theory of gene structure. In a 1948 paper, Norman Horowitz named the concept the "one gene–one enzyme hypothesis". [2]

Although influential, the one gene–one enzyme hypothesis was not unchallenged. Among others, Max Delbrück was skeptical only a single enzyme was actually involved at each step along metabolic pathways. For many who did accept the results, it strengthened the link between genes and enzymes, so that some biochemists thought that genes were enzymes this was consistent with other work, such as studies of the reproduction of tobacco mosaic virus (which was known to have heritable variations and which followed the same pattern of autocatalysis as many enzymatic reactions) and the crystallization of that virus as an apparently pure protein. At the start of the 1950s, the Neurospora findings were widely admired, but the prevailing view in 1951 was that the conclusion Beadle had drawn from them was a vast oversimplification. [8] Beadle wrote in 1966, that after reading the 1951 Cold Spring Harbor Symposium on Genes and Mutations, he had the impression that supporters of the one gene–one enzyme hypothesis “could be counted on the fingers of one hand with a couple of fingers left over.” [10] By the early 1950s, most biochemists and geneticists considered DNA the most likely candidate for physical basis of the gene, and the one gene–one enzyme hypothesis was reinterpreted accordingly. [11]

One gene–one polypeptide Edit

In attributing an instructional role to genes, Beadle and Tatum implicitly accorded genes an informational capability. This insight provided the foundation for the concept of a genetic code. However, it was not until the experiments were performed showing that DNA was the genetic material, that proteins consist of a defined linear sequence of amino acids, and that DNA structure contained a linear sequence of base pairs, was there a clear basis for solving the genetic code.

By the early 1950s, advances in biochemical genetics—spurred in part by the original hypothesis—made the one gene–one enzyme hypothesis seem very unlikely (at least in its original form). Beginning in 1957, Vernon Ingram and others showed through electrophoresis and 2D chromatography that genetic variations in proteins (such as sickle cell hemoglobin) could be limited to differences in just a single polypeptide chain in a multimeric protein, leading to a "one gene–one polypeptide" hypothesis instead. [12] According to geneticist Rowland H. Davis, "By 1958 – indeed, even by 1948 – one gene, one enzyme was no longer a hypothesis to be resolutely defended it was simply the name of a research program." [13]

Presently, the one gene–one polypeptide perspective cannot account for the various spliced versions in many eukaryote organisms which use a spliceosome to individually prepare a RNA transcript depending on the various inter- and intra-cellular environmental signals. This splicing was discovered in 1977 by Phillip Sharp and Richard J. Roberts [14]

Historian Jan Sapp has studied the controversy in regard to German geneticist Franz Moewus who, as some leading geneticists of the 1940s and 50s argued, generated similar results before Beadle and Tatum's celebrated 1941 work. [15] Working on the algae Chlamydomonas, Moewus published, in the 1930s, results that showed that different genes were responsible for different enzymatic reactions in the production of hormones that controlled the organism's reproduction. However, as Sapp skillfully details, those results were challenged by others who found the data 'too good to be true' statistically, and the results could not be replicated.


The chemical biology of modular biosynthetic enzymes

J. L. Meier and M. D. Burkart, Chem. Soc. Rev., 2009, 38, 2012 DOI: 10.1039/B805115C

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.


Faculty and Research Interests

We seek to understand the mechanisms that regulate the balance between stem cell self-renewal and differentiation. We use the germline stem cells of the Drosophila ovary as a model to study stem cell biology because we can identify and genetically manipulate individual cells within their native environment. The molecules we work on tend to fall into two broad categories: Those that regulate chromatin organization and those that modulate mRNA translation. Lab website

Thomas Carroll, Ph.D.

Despite advances in our understanding of the ultrastructure of eukaryotic cells, the question of how these cells interact to form the tissues of our bodies is still poorly understood. Once formed, we know even less about how these tissues are maintained. My lab is interested in how groups of cells organize themselves into properly sized, polarized tubules and then how these structures are maintained throughout the life of the organism. This is particularly significant as defects in tubule size and maintenance play causal roles in several human diseases including cystic kidney diseases and cancers. Lab website

Elizabeth Chen, Ph.D.

Our lab is interested in discovering mechanisms underlying cell-cell fusion, a fascinating process essential for the conception, development, regeneration, and physiology of multicellular organisms. We use genetic, cell biological, biochemical, and biophysical tools to study this process in organisms ranging from insects to mammals. We have to date elucidated the function of the actin cytoskeleton in cell-cell fusion by uncovering an asymmetric fusogenic synapse where mechanical forces are generated to promote cell membrane fusion. Our current effort aims to understand the roles of transmembrane proteins and lipids at the fusogenic synapse. Insights from these studies would provide basis for optimizing therapeutic approaches for tissue degenerative diseases. Lab website

Zhijian &ldquoJames&rdquo Chen, Ph.D.

We are taking biochemical and genetic approaches to dissect two signaling pathways: ubiquitin-mediated activation of protein kinases and antiviral innate immunity. We have uncovered a novel function of ubiquitin in activating protein kinases in the nuclear factor &kappaB (NF-&kappaB) signaling cascade through a proteasome-independent mechanism. Our current effort is focused on elucidating the biochemical mechanism underlying the regulatory function of ubiquitin. In viral signaling, we are particularly interested in dissecting the signaling pathway by which a host cell mounts an immune response to RNA virus infection.

Ondine Cleaver, Ph.D.

During early development, the embryo acquires its shape and complexity of tissues via the coordination of fundamental cellular processes, such as cell signaling, cell migration, cell adhesion and cell differentiation. During this amazing process, a multitude of different signals must be exchanged between cells at precise times and locations, often in a step-wise manner. We are interested in understanding the molecular mechanisms that underlie organogenesis in the embryo. Understanding these principles will help us to identify and characterize molecular lesions that underlie human birth defects and disease. Lab website

Peter Douglas, Ph.D.

Our laboratory is interested in understanding how tissues such as the nervous system respond to traumatic injury. Using high-throughput screening in the nematode C. elegans in combination with murine models, we seek to uncover the short and long-term regenerative capacity for stress response and protein homeostasis pathways in brain injury. Lab website

Jenna Jewell, Ph.D.

The Jewell Lab has a general interest in the regulation of mammalian cell growth and metabolism. Our research is particularly focused on the mechanistic target of rapamycin (mTOR), a highly conserved protein kinase that is implicated in many human diseases. Pharmacological inhibition of mTOR has proven efficacious in several clinical trials and several compounds have been approved by the Food and Drug Administration to treat late-stage renal and breast cancers. mTOR complex 1 (mTORC1) senses multiple stimuli, including growth factors, stress, energy status, and amino acids. Although amino acids are key environmental stimuli, exactly how cells sense them and how they activate mTORC1 is not fully understood. To this end, our lab is interested in elucidating the molecular mechanisms by which mTORC1 senses amino acids to control cellular processes such as growth, autophagy, and metabolism. Lab website

Jin Jiang, Ph.D.

We study genetic pathways and cell signaling mechanisms that regulate embryonic development, adult tissue homeostasis and regeneration. We are particularly interested in studying Hedgehog signaling mechanism in pattern formation, Hippo signaling in organ size control and tumorigenesis, and BMP signaling in stem cell self-renewal and regeneration. Lab website

Steven Kliewer, Ph.D.

Dr. Kliewer runs a joint laboratory with Dr. David Mangelsdorf. Their research focuses on the roles of nuclear receptors and endocrine FGFs in regulating diverse aspects of physiology and pathophysiology, including metabolism and related diseases. Among their ongoing projects, they are studying how FGF19 and FGF21 regulate bile acid and energy homeostasis, respectively, and how the dafachronic acid receptor, DAF-12, regulates the infectious lifecycle of parasitic nematodes. Lab website

Joshua Mendell, M.D., Ph.D.

Our laboratory is interested in mechanisms of post-transcriptional regulation of gene expression and how these pathways influence normal physiology and disease. In particular, we have focused on the regulation and functions of noncoding RNAs with an emphasis on microRNAs (miRNAs). miRNAs are an abundant and diverse family of

20- to 23-nucleotide RNAs that recognize sites of complementarity in target mRNAs, resulting in decay and reduced translation of target transcripts. miRNAs provide important functions in development and physiology and their aberrant activity is associated with several diseases including cancer. Our work on the miRNA pathway focuses mainly on three broad questions: What functions do miRNAs perform in normal physiologic states? How does aberrant miRNA activity contribute to diseases such as cancer? How is miRNA abundance regulated in normal physiology and disease? Lab website

Ping Mu, Ph.D.

We seek to understand the mechanisms of antiandrogen resistance in prostate cancer, to identify novel biomarkers, and to develop therapeutic approaches to prevent or overcome resistance. Lab website

Kathryn O&rsquoDonnell, Ph.D.

The O&rsquoDonnell laboratory is focused on understanding the mechanisms that contribute to tumor initiation, progression, and metastasis. We have identified novel genes that promote liver cancer, leukemia, and non-small cell lung cancer. Currently, we are investigating the regulation and function of cell surface receptors in lung cancer using molecular and biochemical studies, and animal models. These studies may provide new therapeutic approaches to target cancer cells. Lab website

Eric Olson, Ph.D.

Our lab studies muscle cells as a model for understanding how embryonic cells adopt specific fates and how programs of cell differentiation and morphogenesis are controlled during development. There are three major muscle cell types: cardiac, skeletal and smooth, which express distinct sets of genes controlled by different combinations of transcription factors and extracellular signals. We have focused on discovering novel transcription factors that control development of these muscle cell types and remodeling in response to cardiovascular and neuromuscular diseases.

The processes involved in muscle development are evolutionarily ancient and conserved across diverse organisms. This conservation has enabled us to take a cross-species approach to dissect this problem by identifying myogenic regulatory genes in the fruit fly or in vertebrate embryos and to use these genes to perform gain and loss-of-function experiments in vivo and in vitro. Most recently, we have explored the roles of microRNAs in the control of muscle development and disease. Our longterm goal is to delineate the complete genetic pathways for the formation and function of each muscle cell type and to use this information to devise pharmacologic and genetic therapies for inherited and acquired muscle diseases in humans. Lab website

Kim Orth, Ph.D.

My lab is interested in elucidating the activity of virulence factors (also called effectors) from pathogenic bacteria so that we can gain novel molecular insight into eukaryotic signaling systems. We study T3SS systems and bacterial effectors to understand how signaling systems in the eukaryotic host can be manipulated by bacterial pathogens. These studies provide novel insight into the molecular workings of eukaryotic signal transduction. Lab website

Lu Sun, Ph.D.

In vertebrates (including human beings), nearly half of the brain cells are non-neuronal cells. Most of them are so-called &ldquoglial cells&rdquo, which are critical for normal nervous system function. There are three major glial cell types: astrocytes, oligodendrocytes, and microglia. How glial cells develop and how they communicate with neurons and other cell types remain mysterious. Our lab is dedicated to addressing the cellular and molecular mechanisms underlying neuron-glia interactions in health and disease. In particular, we have focused on the oligodendrocytes, the sole myelin-producing cells in the central nervous system, and have recently identified a novel pathway that controls the location and timing of myelination. Our long-term goal aims to elucidate fundamental principles that govern neuron-glia communications. This will help us better characterize many neurological disorders that are greatly contributed by glial dysfunction, including white matter injury, multiple sclerosis, brain tumors, and Alzheimer&rsquos disease. Lab website

Vincent Tagliabracci, Ph.D.

Our lab studies the phosphorylation of extracellular proteins by a novel family of &ldquosecreted kinases.&rdquo This kinase family is so different from canonical kinases that it was not included as a branch on the human kinome tree. The lab uses biochemical and genetic approaches to identify the substrates for these kinases. Current work focuses on one member of this family, Fam20C, which we identified as the bona fide &ldquoGolgi casein kinase&rdquo, an enzyme that escaped identification for many years. Fam20C phosphorylates hundreds of secreted proteins and appears to generate the majority of the extracellular phosphoproteome. Fam20C substrates are involved in a broad spectrum of physiological processes, including lipid metabolism, wound healing, cell migration, biomineralization and inflammation. Understanding the functional implications of these phosphorylation events and how these modifications impact human biology are major objectives of the laboratory. Lab website

Zhigao Wang, Ph.D.

Necrosis is a process clearly distinguished from apoptosis and is implicated in many human pathological conditions, including infections, ischemic injuries, neurodegeneration, and cancer. However, the molecular mechanisms of necrosis remain largely unknown. Using a combination of chemical genetics and biochemical approaches, our research has brought new insights into necrotic cell death pathways, and identified many chemical inhibitors of necrosis that could lead to therapeutic strategies to treat necrosis-related diseases.

Our research goals are to continue investigating the mechanisms of necrotic cell death, especially the executioners of the process, and ultimately to apply this knowledge to develop novel pharmacological interventions to treat necrosis-related human diseases. Activation of the necrotic pathways in tumors that are highly resistant to apoptosis would be a novel direction in cancer treatment.

Jun Wu, Ph.D.

The Wu lab uses interspecies chimeras &ndash entities made up of cells from two different organisms &ndash to study fundamental biology: conserved and divergent developmental programs, determination of body and organ size, species barriers, and cancer resistance. We also work to develop new applications for regenerative medicine. Lab Website

Chun-Li Zhang, Ph.D.

Molecular and cellular biology of postnatal neural stem cells, with a focus on their specification and maintenance. Regeneration of the central nervous system through reprogramming endogenous cells. Molecular signaling in brain tumors. Lab website


Watch the video: Enzymes Updated (August 2022).