- Email: [email protected]
Mechanisms
ch3518.qxd
11/11/1999 9:49 AM
Page 571
571
Mechanisms Research highlights at the chemistry–biology interface Editorial overview Stephen J Benkovic* and Christopher T Walsh† Addresses *The Pennsylvania State University, Department of Chemistry, 152 Davey Laboratory, University Park, PA 16802, USA; e-mail: [email protected] † Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; e-mail: [email protected] Current Opinion in Chemical Biology 1999, 3:571–572 1367-5931/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviation DAP diaminopimelic acid
As genomic information and proteomic information grows exponentially, scientists interested in the function of families of proteins and their recognition of ligands have an almost unlimited opportunity at the biology/chemistry interface to use structure-based approaches, selectionbased approaches, and mechanism-based approaches to delineate the discrete functions of a target protein. Three of the reviews in this issue summarize recent advances in classes of proteins that regulate homeostasis in cells: telomerases; restriction endonucleases; and ATP-dependent proteases. Each of these enzyme classes carry out essential catalytic functions that require temporal coordination in the cells where the enzymes are expressed and as parts of molecular machines. Telomerase, reviewed by Weilbaecher and Lundblad (pp 573–577), synthesizes tandem tracts of a short guanine-rich sequence onto 3′ overhangs of chromosomes, thus compensating for the terminal sequence loss that occurs as a result of DNA replication. For that reason there has been great interest in the role of telomerase in aging and tumorogenesis. The enzyme is a ribonucleoprotein comprising a catalytic protein subunit that is structurally and functionally similar to a reverse transcriptase and an RNA subunit that provides a template for DNA synthesis. A model for the telomerase reaction cycle begins with the binding of a DNA substrate, aligning its 3′ end to that of the RNA template and its 5′ end in an anchor site. Deoxynucleotide addition and translocation proceeds without substrate dissociation, effected by the two binding sites on the telomerase. What isn’t clear is how this cycle may be controlled by other cellular proteins, for example, by blocking telomerase access to the 3′-primer or steps subsequent to primer binding such as processivity or possible proofreading. In the review by Kovall and Matthews (pp 578–583), the hydrolytic mechanisms used by the type II endonucleases
are explored through the enzymes’ X-ray crystal structures. Type II endonucleases are homodimeric enzymes that recognize a palindromic sequence of double-stranded DNA and specifically cleave this sequence in the presence of Mg2+. Members of this family, which includes BamH1, EcoRI, Cfr101, EcoRV, and PvuII, have a structurally similar catalytic core despite little sequence homology at the amino acid level. The catalytic core features three charged residues, typically glutamate or aspartate and lysine. In addition, X-ray crystal structures of three of the enzymes in the presence of Ca2+ are consistent with a mechanism in which the first of two metal ions activates an attacking water molecule, and the second ion stabilizes the leaving group. The universality of this mechanism for DNA cleavage, mismatch repair and synthesis suggests an ancestral protein for all three functions. The review by Schmidt, Lupas and Finley (pp 584–591) provides an overview on the large families of ATP-dependent proteases, which are responsible for the bulk of intracellular protein breakdown and are distributed ubiquitously throughout prokaryotic organisms and in the cytoplasm of eukaryotes. Because proteolysis of peptide bonds is irreversible in vivo, carefully controlled regulation is essential and that is one of the several roles of ATP hydrolysis. The ATP-dependent proteases are machines with self-compartmentalized chambers: only proteins that can be recognized, unfolded, and translocated into the chambers are hydrolyzed. According to Schmidt et al. the regulatory ATPases serve as gatekeepers for controlling access to the proteolytic chambers. In yeast one of the regulatory subassemblies of the proteasome has six ATPase subunits, each with nonredundant function, suggesting many aspects of chaperoning, unfolding, and control of coupled equilibria are at work to coordinate intracellular proteolysis. Four of the reviews in this issue deal with different aspects of the biology of antibiotics or their targets. The two chapters by Floss and Yu (pp 592–597) and by Keating and Walsh (pp 598–606) set perspectives on several of the recent developments in the assembly of antibiotics by multimodular catalysts that make polyketides and nonribosomal polypeptides. The Keating and Walsh review dissects the cascades of acyl enzyme intermediates into steps involved in acyl chain initiation, elongation, and termination. Initiation is the strategy for selection, activation, and covalent loading of the monomer units to be assembled into the natural products. Elongation events deal with the condensation of an upstream acyl chain on to downstream ones as the growing chain translocates between
ch3518.qxd
11/11/1999 9:49 AM
572
Page 572
Mechanisms
carrier protein domains, as well as chemical modifications to the chains that occur within an elongation cycle. Termination requires that the full length acyl chain be disconnected from its covalent niche on the polyketide synthase or nonribosomal peptide synthetase, generally a hydrolytic step. The Floss and Yu review deals with the rifamycin class of macrocyclic polyketides that have a central role in antituberculosis therapeutics. Recent lessons that have illuminated this area of natural product enzymology include: the use of an unusual starter group, the aminohydroxybenzoate group; the demonstration that the acyl chain termination step is catalyzed by a separate amide synthetase (RifB); and that a whole assembly line of acyl chains stalls and builds up in a RifB mutant. In addition, evidence that napthoquinone ring closure can be cosynthetic with chain elongation is presented. All these lessons complement, augment, and revise some of the large body of information on the templating and organization of type I polyketide synthases that has been provided by the paradigm in this field, the 6-deoxyerythronolide B synthase. The peptidoglycan layer of the bacterial cell wall and the enzymes that provide the components and assemble them into peptidoglycan have long been important targets of antibiotics, including the β-lacatms (penicillins and cephalosporins) and vancomycin. A crucial component in the peptide portion of peptidoglycan is the double-headed amino acid diaminopimelic acid (DAP), which provides the C6 amino group that functions as the nucleophile in cross-linking of one peptide strand to another in the peptidoglycan meshwork. Whereas the nine enzymes that convert aspartate to DAP have been identified, only recently has there been an explosion of structural and attendant functional information, as summarized by Born and Blanchard (pp 607–613). This new information includes the structure determinations of tetrahydrodipicolinate N-succinyltransferase, the DAP epimerase and the meso-DAP dehydrogenase as well as detailed mechanistic studies on the succinyl–DAP aminotransferase and desuccinylase. In aggregate this information should enhance the ability to screen and/or design for selective, potent inhibitors of these enzymes and block bacterial cell wall biosynthesis. When β-lactam-based antibiotic therapy fails in a patient, the cause is often that the resistant bacteria are overproducing a β-lactamase that hydrolyzes the penicillin or cephalosporin in the bacterial periplasm before the drug can run the gauntlet intact and reach its targets, the peptidoglycan cross-linking enzymes in the cell membrane of the bacteria. Four main classes of lactamases have been defined (A–D), of which classes A, C and D use active site serine residues as catalytic nucleophiles in C–N bond cleavage of the β-lactam. Class B β-lactamases are differ-
ent, in requiring an active site metal ion for catalytic activity. This has important practical consequences since the class A, C and D lactamases are targetted by the combination drugs that add a mechanism-based inactivator (e.g. clavulanate or sulbactam) to penicillins and thereby allow penicillins to avoid hydrolytic destruction. The class B metallo-β-lactamases are not susceptible to this approach and require distinct targetting methods. For this reason research into the mechanism of the monozinc and bizinc β-lactamases is particularly timely and welcome. Wang et al. (pp 614–622) review both new structural evidence on the monozinc lacatamase from Bacillus cereus and the dizinc enzyme from Bacillus fragilis and elucidate the evolution of catalytic mechanisms for this enzyme subfamily. The review by Begley and coworkers (pp 623–629) reminds us of the recent breakthroughs in understanding of how C–S bonds are fashioned in the biogenesis of such key small molecules in metabolism as the coenzymes biotin, thiamin, lipoate and molybdopterins. New paradigms for enzyme chemistry have arisen from both the decades long search for the molecular logic of the thiazole ring in thiamin biosynthesis and also the thiolane ring in biotin assembly. In the thiamin case a carboxy-terminal thiocarboxylate in the ThiS protein has been unambiguously identified by elegant electrospray/Fourier transform mass spectroscopic methods. The ThiS thiocarboxylate arises from a ThiS–acylAMP intermediate, which has parallels in the activation of ubiquitin in the proteasome pathway. The dethiobiotin to biotin sulfur insertion is even more baroque; the Fe/S cluster of the biotin synthase is the sulfur donor, thus the enzyme acts as a reagent rather than a catalyst, until the Fe/S cluster is reconstituted by a sulfur insertase. The sulfur–carbon bond insertion involves radical chemistry, initiated by S-adenosylmethronine fragmentation, a radical initiating step now seen in five distinct enzyme systems that make low potential radicals. These ancient vitamins, essential to life, probably evolved in anaerobic settings and have preserved the C–S bond-forming machinery that evolved in those oxygenfree microenvironments. These chapters also illustrate nature’s strategies for dealing with complex chemical syntheses (coenzymes, antibiotics, polyketides) and with the degradation of hydrolytically stable substrates. Complex synthesis requiring regio-control and stereo-control are elaborated through a series of intermediates each fashioned at its own active site, probably within clusters of associated enzymes. Hydrolytically stable linkages exemplified by phosphate and amide bonds are weakened through the strong electrostatic forces emanating from juxtaposed metal ions, often two or three in number. The theme of machine-like complexes repeats itself in the molecular design of telomerases and proteasomes, enzymes which have the unenviable but vital tasks of tidying up.
11/11/1999 9:49 AM
Page 571
571
Mechanisms Research highlights at the chemistry–biology interface Editorial overview Stephen J Benkovic* and Christopher T Walsh† Addresses *The Pennsylvania State University, Department of Chemistry, 152 Davey Laboratory, University Park, PA 16802, USA; e-mail: [email protected] † Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; e-mail: [email protected] Current Opinion in Chemical Biology 1999, 3:571–572 1367-5931/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviation DAP diaminopimelic acid
As genomic information and proteomic information grows exponentially, scientists interested in the function of families of proteins and their recognition of ligands have an almost unlimited opportunity at the biology/chemistry interface to use structure-based approaches, selectionbased approaches, and mechanism-based approaches to delineate the discrete functions of a target protein. Three of the reviews in this issue summarize recent advances in classes of proteins that regulate homeostasis in cells: telomerases; restriction endonucleases; and ATP-dependent proteases. Each of these enzyme classes carry out essential catalytic functions that require temporal coordination in the cells where the enzymes are expressed and as parts of molecular machines. Telomerase, reviewed by Weilbaecher and Lundblad (pp 573–577), synthesizes tandem tracts of a short guanine-rich sequence onto 3′ overhangs of chromosomes, thus compensating for the terminal sequence loss that occurs as a result of DNA replication. For that reason there has been great interest in the role of telomerase in aging and tumorogenesis. The enzyme is a ribonucleoprotein comprising a catalytic protein subunit that is structurally and functionally similar to a reverse transcriptase and an RNA subunit that provides a template for DNA synthesis. A model for the telomerase reaction cycle begins with the binding of a DNA substrate, aligning its 3′ end to that of the RNA template and its 5′ end in an anchor site. Deoxynucleotide addition and translocation proceeds without substrate dissociation, effected by the two binding sites on the telomerase. What isn’t clear is how this cycle may be controlled by other cellular proteins, for example, by blocking telomerase access to the 3′-primer or steps subsequent to primer binding such as processivity or possible proofreading. In the review by Kovall and Matthews (pp 578–583), the hydrolytic mechanisms used by the type II endonucleases
are explored through the enzymes’ X-ray crystal structures. Type II endonucleases are homodimeric enzymes that recognize a palindromic sequence of double-stranded DNA and specifically cleave this sequence in the presence of Mg2+. Members of this family, which includes BamH1, EcoRI, Cfr101, EcoRV, and PvuII, have a structurally similar catalytic core despite little sequence homology at the amino acid level. The catalytic core features three charged residues, typically glutamate or aspartate and lysine. In addition, X-ray crystal structures of three of the enzymes in the presence of Ca2+ are consistent with a mechanism in which the first of two metal ions activates an attacking water molecule, and the second ion stabilizes the leaving group. The universality of this mechanism for DNA cleavage, mismatch repair and synthesis suggests an ancestral protein for all three functions. The review by Schmidt, Lupas and Finley (pp 584–591) provides an overview on the large families of ATP-dependent proteases, which are responsible for the bulk of intracellular protein breakdown and are distributed ubiquitously throughout prokaryotic organisms and in the cytoplasm of eukaryotes. Because proteolysis of peptide bonds is irreversible in vivo, carefully controlled regulation is essential and that is one of the several roles of ATP hydrolysis. The ATP-dependent proteases are machines with self-compartmentalized chambers: only proteins that can be recognized, unfolded, and translocated into the chambers are hydrolyzed. According to Schmidt et al. the regulatory ATPases serve as gatekeepers for controlling access to the proteolytic chambers. In yeast one of the regulatory subassemblies of the proteasome has six ATPase subunits, each with nonredundant function, suggesting many aspects of chaperoning, unfolding, and control of coupled equilibria are at work to coordinate intracellular proteolysis. Four of the reviews in this issue deal with different aspects of the biology of antibiotics or their targets. The two chapters by Floss and Yu (pp 592–597) and by Keating and Walsh (pp 598–606) set perspectives on several of the recent developments in the assembly of antibiotics by multimodular catalysts that make polyketides and nonribosomal polypeptides. The Keating and Walsh review dissects the cascades of acyl enzyme intermediates into steps involved in acyl chain initiation, elongation, and termination. Initiation is the strategy for selection, activation, and covalent loading of the monomer units to be assembled into the natural products. Elongation events deal with the condensation of an upstream acyl chain on to downstream ones as the growing chain translocates between
ch3518.qxd
11/11/1999 9:49 AM
572
Page 572
Mechanisms
carrier protein domains, as well as chemical modifications to the chains that occur within an elongation cycle. Termination requires that the full length acyl chain be disconnected from its covalent niche on the polyketide synthase or nonribosomal peptide synthetase, generally a hydrolytic step. The Floss and Yu review deals with the rifamycin class of macrocyclic polyketides that have a central role in antituberculosis therapeutics. Recent lessons that have illuminated this area of natural product enzymology include: the use of an unusual starter group, the aminohydroxybenzoate group; the demonstration that the acyl chain termination step is catalyzed by a separate amide synthetase (RifB); and that a whole assembly line of acyl chains stalls and builds up in a RifB mutant. In addition, evidence that napthoquinone ring closure can be cosynthetic with chain elongation is presented. All these lessons complement, augment, and revise some of the large body of information on the templating and organization of type I polyketide synthases that has been provided by the paradigm in this field, the 6-deoxyerythronolide B synthase. The peptidoglycan layer of the bacterial cell wall and the enzymes that provide the components and assemble them into peptidoglycan have long been important targets of antibiotics, including the β-lacatms (penicillins and cephalosporins) and vancomycin. A crucial component in the peptide portion of peptidoglycan is the double-headed amino acid diaminopimelic acid (DAP), which provides the C6 amino group that functions as the nucleophile in cross-linking of one peptide strand to another in the peptidoglycan meshwork. Whereas the nine enzymes that convert aspartate to DAP have been identified, only recently has there been an explosion of structural and attendant functional information, as summarized by Born and Blanchard (pp 607–613). This new information includes the structure determinations of tetrahydrodipicolinate N-succinyltransferase, the DAP epimerase and the meso-DAP dehydrogenase as well as detailed mechanistic studies on the succinyl–DAP aminotransferase and desuccinylase. In aggregate this information should enhance the ability to screen and/or design for selective, potent inhibitors of these enzymes and block bacterial cell wall biosynthesis. When β-lactam-based antibiotic therapy fails in a patient, the cause is often that the resistant bacteria are overproducing a β-lactamase that hydrolyzes the penicillin or cephalosporin in the bacterial periplasm before the drug can run the gauntlet intact and reach its targets, the peptidoglycan cross-linking enzymes in the cell membrane of the bacteria. Four main classes of lactamases have been defined (A–D), of which classes A, C and D use active site serine residues as catalytic nucleophiles in C–N bond cleavage of the β-lactam. Class B β-lactamases are differ-
ent, in requiring an active site metal ion for catalytic activity. This has important practical consequences since the class A, C and D lactamases are targetted by the combination drugs that add a mechanism-based inactivator (e.g. clavulanate or sulbactam) to penicillins and thereby allow penicillins to avoid hydrolytic destruction. The class B metallo-β-lactamases are not susceptible to this approach and require distinct targetting methods. For this reason research into the mechanism of the monozinc and bizinc β-lactamases is particularly timely and welcome. Wang et al. (pp 614–622) review both new structural evidence on the monozinc lacatamase from Bacillus cereus and the dizinc enzyme from Bacillus fragilis and elucidate the evolution of catalytic mechanisms for this enzyme subfamily. The review by Begley and coworkers (pp 623–629) reminds us of the recent breakthroughs in understanding of how C–S bonds are fashioned in the biogenesis of such key small molecules in metabolism as the coenzymes biotin, thiamin, lipoate and molybdopterins. New paradigms for enzyme chemistry have arisen from both the decades long search for the molecular logic of the thiazole ring in thiamin biosynthesis and also the thiolane ring in biotin assembly. In the thiamin case a carboxy-terminal thiocarboxylate in the ThiS protein has been unambiguously identified by elegant electrospray/Fourier transform mass spectroscopic methods. The ThiS thiocarboxylate arises from a ThiS–acylAMP intermediate, which has parallels in the activation of ubiquitin in the proteasome pathway. The dethiobiotin to biotin sulfur insertion is even more baroque; the Fe/S cluster of the biotin synthase is the sulfur donor, thus the enzyme acts as a reagent rather than a catalyst, until the Fe/S cluster is reconstituted by a sulfur insertase. The sulfur–carbon bond insertion involves radical chemistry, initiated by S-adenosylmethronine fragmentation, a radical initiating step now seen in five distinct enzyme systems that make low potential radicals. These ancient vitamins, essential to life, probably evolved in anaerobic settings and have preserved the C–S bond-forming machinery that evolved in those oxygenfree microenvironments. These chapters also illustrate nature’s strategies for dealing with complex chemical syntheses (coenzymes, antibiotics, polyketides) and with the degradation of hydrolytically stable substrates. Complex synthesis requiring regio-control and stereo-control are elaborated through a series of intermediates each fashioned at its own active site, probably within clusters of associated enzymes. Hydrolytically stable linkages exemplified by phosphate and amide bonds are weakened through the strong electrostatic forces emanating from juxtaposed metal ions, often two or three in number. The theme of machine-like complexes repeats itself in the molecular design of telomerases and proteasomes, enzymes which have the unenviable but vital tasks of tidying up.