Christopher Thibodeaux | University of Illinois at Urbana-Champaign (original) (raw)

Papers by Christopher Thibodeaux

Journal of the American Chemical Society, Jan 28, 2016

The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multip... more The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multiple chemical processes are poorly understood. The lacticin 481 synthetase (LctM) cleaves eight chemical bonds and forms six new chemical bonds in a controlled and ordered process. Two general mechanisms have been suggested for the temporal and spatial control of these transformations. In the spatial positioning model, leader peptide binding promotes certain reactions by establishing the spatial orientation of the substrate peptide relative to the synthetase active sites. In the intermediate structure model, the LctM-catalyzed dehydration and cyclization reactions that occur in two distinct active sites orchestrate the overall process by imparting structure into the maturing peptide. Using isotopically labeled LctA analogues with engineered lacticin 481 biosynthetic machinery and mass spectrometry analysis, we show here that the LctA leader peptide plays critical roles in establishing the ...

Proceedings of the National Academy of Sciences of the United States of America, Jan 31, 2015

Although natural products have been a particularly rich source of human medicines, activity-based... more Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the dis...

Biochemistry, 2011

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-... more 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give R-ketobutyric acid and ammonia as products. The cleavage of the C R -C β bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile-or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK a of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and 13 C KIE studies of the wild type enzyme suggest that the C R -C β bond cleavage step in the chemical mechanism is at least partially rate-limiting under k cat /K m conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294. FIGURE 1: Active site of wild type ACCD from Pseudomonas sp.

Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide na... more Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) that typically harbor multiple intramolecular thioether linkages. For class II lanthipeptides, these crosslinks are installed in a multistep reaction pathway by a single enzyme (LanM). The multi-functional nature of LanMs and the manipulability of their genetically encoded peptide substrates (LanAs) make LanM/LanA systems promising targets for the engineering of new antibacterial compounds. Here, we report the development of a semi-quantitative mass spectrometry-based assay for kinetic characterization of LanMcatalyzed reactions. The assay was used to conduct a comparative kinetic analysis of two LanM enzymes (HalM2 and ProcM) that exhibit drastically different substrate selectivity. Numerical simulation of the kinetic data was used to develop models for the multistep HalM2-and ProcM-catalyzed reactions. These models illustrate that HalM2 and ProcM have markedly different catalytic efficiencies for the various reactions they catalyze. HalM2, which is responsible for the biosynthesis of a single compound (the Halβ subunit of the lantibiotic haloduracin), catalyzes reactions with higher catalytic efficiency than ProcM, which modifies 29 different ProcA precursor peptides during prochlorosin biosynthesis. In particular, the rates of thioether ring formation are drastically reduced in ProcM, likely because this enzyme is charged with installing a variety of lanthipeptide ring architectures in its prochlorosin products. Thus, ProcM appears to pay a kinetic price for its relaxed substrate specificity. In addition, our kinetic models suggest that conformational sampling of the LanM:LanA Michaelis complex could play an important role in the kinetics of LanA maturation. at 1.12 and 1.14 the ߯ ଶ minimum, respectively. These thresholds provide a larger boundary range for the parameter estimates than the thresholds suggested by FitSpace Explorer (1.06 and 1.07, respectively).

Journal of the American …, Jan 1, 2010

The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the rev... more The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible isomerization of the two ubiquitous isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are required to initiate the biosynthesis of all isoprenoid compounds found in nature. The overall chemical transformation catalyzed by IDI-2 involves a net 1,3-proton addition/elimination reaction. Surprisingly, IDI-2 requires a reduced flavin mononucleotide (FMN) coenzyme to carry out this redox neutral isomerization. The exact function of FMN in catalysis has not yet been clearly defined. To provide mechanistic insight into the role of the reduced flavin in IDI-2 catalysis, several FMN analogues with altered electronic properties were chemoenzymatically prepared, and their effects on the kinetic properties of the IDI-2 catalyzed reaction were investigated. Linear free energy relationships (LFERs) between the electronic properties of the flavin and the steady state kinetic parameters of the IDI-2 catalyzed reaction were observed. The LFER studies are complemented with kinetic isotope effect studies and kinetic characterization of an active site mutant enzyme (Q154N). Cumulatively, the data presented in this work (and in other studies) suggest that the reduced FMN coenzyme of IDI-2 functions as an acid/base catalyst, with the N5 atom of the flavin likely playing a critical role in the deprotonation of IPP en route to DMAPP formation. Several potential chemical mechanisms involving the reduced flavin as an acid/base catalyst are presented and discussed. Scheme 1.

Biochemistry, Jan 1, 2008

The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) i... more The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O 2 -sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies appear to support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.

Biochemistry, Jan 1, 2011

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-... more 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give R-ketobutyric acid and ammonia as products. The cleavage of the C R -C β bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile-or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK a of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and 13 C KIE studies of the wild type enzyme suggest that the C R -C β bond cleavage step in the chemical mechanism is at least partially rate-limiting under k cat /K m conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294. FIGURE 1: Active site of wild type ACCD from Pseudomonas sp.

…, Jan 1, 2012

The interconversion of isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2) ... more The interconversion of isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2) by isopentenyl diphosphate-dimethylallyl diphosphate isomerase (IDI) is a key reaction in the synthesis of the building blocks for isoprenoid compounds. Two structurally unrelated types of IDI have been identified. The type I enzyme (IDI-1) is a zinc metalloprotein that also requires Mg 2 + for activity. It employs appropriate active site amino acid residues as general acid and base catalysts to carry out the isomerization reaction. [2g, 3] In contrast, the type II IDI (IDI-2), discovered in 2001, is a flavoprotein that requires a reduced flavin mononucleotide (FMN red , 3) coenzyme in addition to Mg 2 + for activity. [5] The reaction catalyzed by IDI-2 is unusual in that it utilizes the redox active FMN coenzyme to perform a reaction that does not involve a change in the redox state of the substrate/product. This observation raises questions as to the exact role of the flavin coenzyme in the catalytic mechanism. In addition, several human pathogens, such as Staphylococcus aureus, rely exclusively on IDI-2 for the initiation of long chain isoprenoid biosynthesis, whereas in humans the structurally unrelated IDI-1 is employed. Thus, IDI-2 is a potential target for new antimicrobial agents.

Biochemistry, Jan 1, 2007

The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) i... more The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2 sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies of S. aureus IDI-2 support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.

Journal of Biological …, Jan 1, 2012

Background: UDP-galactopyranose mutase (UGM) requires the reduced FAD coenzyme to interconvert UD... more Background: UDP-galactopyranose mutase (UGM) requires the reduced FAD coenzyme to interconvert UDP-galactopyranose and UDP-galactofuranose. Results: Structural perturbations of the coenzyme inhibit bond cleavage in the substrate. Conclusion: Concerted bond breaking and formation between substrate and coenzyme occur during UGM catalysis. Significance: Mechanistic understanding of UGM offers new insight for clinically relevant inhibitor design.

Chemical reviews, Jan 1, 2011

Angewandte Chemie …, Jan 1, 2008

The Journal of organic …, Jan 1, 2007

Flavin coenzymes play a variety of roles in biological systems. This Perspective highlights the c... more Flavin coenzymes play a variety of roles in biological systems. This Perspective highlights the chemical versatility of flavins by reviewing research on five flavoenzymes that have been studied in our laboratory. Each of the enzymes discussed in this review (the acyl-CoA dehydrogenases (ACDs), CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), CDP-4-aceto-3,6-dideoxygalactose synthase (YerE), UDP-galactopyranose mutase (UGM), and type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2)) utilize flavin in a distinct role. In particular, the catalytic mechanisms of two of these enzymes, UGM and IDI-2, may involve novel flavin chemistry.

CHIMIA International Journal for …, Jan 1, 2009

Isoprenoids (or terpenoids) are a large and structurally diverse class of biomolecules that are e... more Isoprenoids (or terpenoids) are a large and structurally diverse class of biomolecules that are essential for the survival of all forms of life. Despite the vast differences in their final structures and functions, the early steps of isoprenoid biosynthesis in all organisms follow one of only two known biosynthetic pathways: the mevalonate pathway or the methyl erythritol phosphate (MEP) pathway. Interestingly, while humans utilize the mevalonate pathway, many human pathogens rely exclusively on the MEP pathway for the biosynthesis of their isoprenoid compounds. This has led to a number of mechanistic studies of the MEP-specific pathway enzymes, with the ultimate goal of developing small molecule inhibitors as potential drugs. In addition to their therapeutic value, many of the MEP pathway enzymes also catalyze unusual chemical transformations that are not well understood. In this review, we will highlight the recent work by us and others towards the elucidation of the catalytic mechanisms of several key enzymes involved in the early stages of isoprenoid biosynthesis. These include 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH) of the MEP pathway, and the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) from Staphylococcus aureus. The functions of these enzymes are validated or identified as potential drug targets.

ACS chemical biology, Jan 1, 2006

Nature, Jan 1, 2007

The enzymes involved in the biosynthesis of carbohydrates and the attachment of sugar units to bi... more The enzymes involved in the biosynthesis of carbohydrates and the attachment of sugar units to biological acceptor molecules catalyse an array of chemical transformations and coupling reactions. In prokaryotes, both common sugar precursors and their enzymatically modified derivatives often become substituents of biologically active natural products through the action of glycosyltransferases. Recently, researchers have begun to harness the power of these biological catalysts to alter the sugar structures and glycosylation patterns of natural products both in vivo and in vitro. Biochemical and structural studies of sugar biosynthetic enzymes and glycosyltransferases, coupled with advances in bioengineering methodology, have ushered in a new era of drug development.

Pure and applied chemistry, Jan 1, 2007

Changing the sugar structures and glycosylation patterns of natural products is an effective mean... more Changing the sugar structures and glycosylation patterns of natural products is an effective means of altering the biological activity of clinically useful drugs. Several recent strategies have provided researchers with the opportunity to manipulate sugar structures and to change the sugar moieties attached to these natural products via a biosynthetic approach. In this review, we explore the utility of contemporary in vivo and in vitro methods to achieve natural product glycodiversification. This study will focus on recent progress from our laboratory in elucidating the biosynthesis of D-desosamine, a deoxysugar component of many macrolide antibiotics, and will highlight how we have engineered the D-desosamine biosynthetic pathway in Streptomyces venezuelae through targeted disruption and heterologous expression of the sugar biosynthetic genes to generate a variety of new glycoforms. The in vitro exploitation of the substrate flexibility of the endogenous D-desosamine glycosyltransferase (GT) to generate many non-natural glycoforms will also be discussed. These experiments are compared with recent work from other research groups on the same topics. Finally, the significance of these studies for the future prospects of natural product glycodiversification is discussed. © 2007 IUPAC, Pure and Applied Chemistry 79, 785-799 786

Methods in enzymology, Jan 1, 2009

Angewandte Chemie, Jan 1, 2008

Journal of the American Chemical Society, Jan 28, 2016

The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multip... more The mechanisms by which lanthipeptide synthetases control the order in which they catalyze multiple chemical processes are poorly understood. The lacticin 481 synthetase (LctM) cleaves eight chemical bonds and forms six new chemical bonds in a controlled and ordered process. Two general mechanisms have been suggested for the temporal and spatial control of these transformations. In the spatial positioning model, leader peptide binding promotes certain reactions by establishing the spatial orientation of the substrate peptide relative to the synthetase active sites. In the intermediate structure model, the LctM-catalyzed dehydration and cyclization reactions that occur in two distinct active sites orchestrate the overall process by imparting structure into the maturing peptide. Using isotopically labeled LctA analogues with engineered lacticin 481 biosynthetic machinery and mass spectrometry analysis, we show here that the LctA leader peptide plays critical roles in establishing the ...

Proceedings of the National Academy of Sciences of the United States of America, Jan 31, 2015

Although natural products have been a particularly rich source of human medicines, activity-based... more Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the dis...

Biochemistry, 2011

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-... more 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give R-ketobutyric acid and ammonia as products. The cleavage of the C R -C β bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile-or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK a of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and 13 C KIE studies of the wild type enzyme suggest that the C R -C β bond cleavage step in the chemical mechanism is at least partially rate-limiting under k cat /K m conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294. FIGURE 1: Active site of wild type ACCD from Pseudomonas sp.

Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide na... more Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) that typically harbor multiple intramolecular thioether linkages. For class II lanthipeptides, these crosslinks are installed in a multistep reaction pathway by a single enzyme (LanM). The multi-functional nature of LanMs and the manipulability of their genetically encoded peptide substrates (LanAs) make LanM/LanA systems promising targets for the engineering of new antibacterial compounds. Here, we report the development of a semi-quantitative mass spectrometry-based assay for kinetic characterization of LanMcatalyzed reactions. The assay was used to conduct a comparative kinetic analysis of two LanM enzymes (HalM2 and ProcM) that exhibit drastically different substrate selectivity. Numerical simulation of the kinetic data was used to develop models for the multistep HalM2-and ProcM-catalyzed reactions. These models illustrate that HalM2 and ProcM have markedly different catalytic efficiencies for the various reactions they catalyze. HalM2, which is responsible for the biosynthesis of a single compound (the Halβ subunit of the lantibiotic haloduracin), catalyzes reactions with higher catalytic efficiency than ProcM, which modifies 29 different ProcA precursor peptides during prochlorosin biosynthesis. In particular, the rates of thioether ring formation are drastically reduced in ProcM, likely because this enzyme is charged with installing a variety of lanthipeptide ring architectures in its prochlorosin products. Thus, ProcM appears to pay a kinetic price for its relaxed substrate specificity. In addition, our kinetic models suggest that conformational sampling of the LanM:LanA Michaelis complex could play an important role in the kinetics of LanA maturation. at 1.12 and 1.14 the ߯ ଶ minimum, respectively. These thresholds provide a larger boundary range for the parameter estimates than the thresholds suggested by FitSpace Explorer (1.06 and 1.07, respectively).

Journal of the American …, Jan 1, 2010

The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the rev... more The type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible isomerization of the two ubiquitous isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are required to initiate the biosynthesis of all isoprenoid compounds found in nature. The overall chemical transformation catalyzed by IDI-2 involves a net 1,3-proton addition/elimination reaction. Surprisingly, IDI-2 requires a reduced flavin mononucleotide (FMN) coenzyme to carry out this redox neutral isomerization. The exact function of FMN in catalysis has not yet been clearly defined. To provide mechanistic insight into the role of the reduced flavin in IDI-2 catalysis, several FMN analogues with altered electronic properties were chemoenzymatically prepared, and their effects on the kinetic properties of the IDI-2 catalyzed reaction were investigated. Linear free energy relationships (LFERs) between the electronic properties of the flavin and the steady state kinetic parameters of the IDI-2 catalyzed reaction were observed. The LFER studies are complemented with kinetic isotope effect studies and kinetic characterization of an active site mutant enzyme (Q154N). Cumulatively, the data presented in this work (and in other studies) suggest that the reduced FMN coenzyme of IDI-2 functions as an acid/base catalyst, with the N5 atom of the flavin likely playing a critical role in the deprotonation of IPP en route to DMAPP formation. Several potential chemical mechanisms involving the reduced flavin as an acid/base catalyst are presented and discussed. Scheme 1.

Biochemistry, Jan 1, 2008

The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) i... more The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O 2 -sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies appear to support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.

Biochemistry, Jan 1, 2011

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-... more 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5 0 -phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give R-ketobutyric acid and ammonia as products. The cleavage of the C R -C β bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile-or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK a of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and 13 C KIE studies of the wild type enzyme suggest that the C R -C β bond cleavage step in the chemical mechanism is at least partially rate-limiting under k cat /K m conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294. FIGURE 1: Active site of wild type ACCD from Pseudomonas sp.

…, Jan 1, 2012

The interconversion of isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2) ... more The interconversion of isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2) by isopentenyl diphosphate-dimethylallyl diphosphate isomerase (IDI) is a key reaction in the synthesis of the building blocks for isoprenoid compounds. Two structurally unrelated types of IDI have been identified. The type I enzyme (IDI-1) is a zinc metalloprotein that also requires Mg 2 + for activity. It employs appropriate active site amino acid residues as general acid and base catalysts to carry out the isomerization reaction. [2g, 3] In contrast, the type II IDI (IDI-2), discovered in 2001, is a flavoprotein that requires a reduced flavin mononucleotide (FMN red , 3) coenzyme in addition to Mg 2 + for activity. [5] The reaction catalyzed by IDI-2 is unusual in that it utilizes the redox active FMN coenzyme to perform a reaction that does not involve a change in the redox state of the substrate/product. This observation raises questions as to the exact role of the flavin coenzyme in the catalytic mechanism. In addition, several human pathogens, such as Staphylococcus aureus, rely exclusively on IDI-2 for the initiation of long chain isoprenoid biosynthesis, whereas in humans the structurally unrelated IDI-1 is employed. Thus, IDI-2 is a potential target for new antimicrobial agents.

Biochemistry, Jan 1, 2007

The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) i... more The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2 sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation-reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies of S. aureus IDI-2 support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.

Journal of Biological …, Jan 1, 2012

Background: UDP-galactopyranose mutase (UGM) requires the reduced FAD coenzyme to interconvert UD... more Background: UDP-galactopyranose mutase (UGM) requires the reduced FAD coenzyme to interconvert UDP-galactopyranose and UDP-galactofuranose. Results: Structural perturbations of the coenzyme inhibit bond cleavage in the substrate. Conclusion: Concerted bond breaking and formation between substrate and coenzyme occur during UGM catalysis. Significance: Mechanistic understanding of UGM offers new insight for clinically relevant inhibitor design.

Chemical reviews, Jan 1, 2011

Angewandte Chemie …, Jan 1, 2008

The Journal of organic …, Jan 1, 2007

Flavin coenzymes play a variety of roles in biological systems. This Perspective highlights the c... more Flavin coenzymes play a variety of roles in biological systems. This Perspective highlights the chemical versatility of flavins by reviewing research on five flavoenzymes that have been studied in our laboratory. Each of the enzymes discussed in this review (the acyl-CoA dehydrogenases (ACDs), CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), CDP-4-aceto-3,6-dideoxygalactose synthase (YerE), UDP-galactopyranose mutase (UGM), and type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2)) utilize flavin in a distinct role. In particular, the catalytic mechanisms of two of these enzymes, UGM and IDI-2, may involve novel flavin chemistry.

CHIMIA International Journal for …, Jan 1, 2009

Isoprenoids (or terpenoids) are a large and structurally diverse class of biomolecules that are e... more Isoprenoids (or terpenoids) are a large and structurally diverse class of biomolecules that are essential for the survival of all forms of life. Despite the vast differences in their final structures and functions, the early steps of isoprenoid biosynthesis in all organisms follow one of only two known biosynthetic pathways: the mevalonate pathway or the methyl erythritol phosphate (MEP) pathway. Interestingly, while humans utilize the mevalonate pathway, many human pathogens rely exclusively on the MEP pathway for the biosynthesis of their isoprenoid compounds. This has led to a number of mechanistic studies of the MEP-specific pathway enzymes, with the ultimate goal of developing small molecule inhibitors as potential drugs. In addition to their therapeutic value, many of the MEP pathway enzymes also catalyze unusual chemical transformations that are not well understood. In this review, we will highlight the recent work by us and others towards the elucidation of the catalytic mechanisms of several key enzymes involved in the early stages of isoprenoid biosynthesis. These include 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH) of the MEP pathway, and the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) from Staphylococcus aureus. The functions of these enzymes are validated or identified as potential drug targets.

ACS chemical biology, Jan 1, 2006

Nature, Jan 1, 2007

The enzymes involved in the biosynthesis of carbohydrates and the attachment of sugar units to bi... more The enzymes involved in the biosynthesis of carbohydrates and the attachment of sugar units to biological acceptor molecules catalyse an array of chemical transformations and coupling reactions. In prokaryotes, both common sugar precursors and their enzymatically modified derivatives often become substituents of biologically active natural products through the action of glycosyltransferases. Recently, researchers have begun to harness the power of these biological catalysts to alter the sugar structures and glycosylation patterns of natural products both in vivo and in vitro. Biochemical and structural studies of sugar biosynthetic enzymes and glycosyltransferases, coupled with advances in bioengineering methodology, have ushered in a new era of drug development.

Pure and applied chemistry, Jan 1, 2007

Changing the sugar structures and glycosylation patterns of natural products is an effective mean... more Changing the sugar structures and glycosylation patterns of natural products is an effective means of altering the biological activity of clinically useful drugs. Several recent strategies have provided researchers with the opportunity to manipulate sugar structures and to change the sugar moieties attached to these natural products via a biosynthetic approach. In this review, we explore the utility of contemporary in vivo and in vitro methods to achieve natural product glycodiversification. This study will focus on recent progress from our laboratory in elucidating the biosynthesis of D-desosamine, a deoxysugar component of many macrolide antibiotics, and will highlight how we have engineered the D-desosamine biosynthetic pathway in Streptomyces venezuelae through targeted disruption and heterologous expression of the sugar biosynthetic genes to generate a variety of new glycoforms. The in vitro exploitation of the substrate flexibility of the endogenous D-desosamine glycosyltransferase (GT) to generate many non-natural glycoforms will also be discussed. These experiments are compared with recent work from other research groups on the same topics. Finally, the significance of these studies for the future prospects of natural product glycodiversification is discussed. © 2007 IUPAC, Pure and Applied Chemistry 79, 785-799 786

Methods in enzymology, Jan 1, 2009

Angewandte Chemie, Jan 1, 2008