steven van lanen | University of Kentucky (original) (raw)

Papers by steven van lanen

Applied and Environmental Microbiology, 2015

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyce... more Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result many attempts have been made to develop ABM into a drug in the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfuramino acid (saa) metabolism that supplies a precursor for ABM biosynthesis could lead to an improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus, and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). Gene product AbmD appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplement in growth media increased ABM production by significantly changing relative concentrations of intracellular Cys and Hcy. A saa metabolic network of S. griseus was constructed. Pathways towards increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. ABM titer of one engineered strain SCAK3 in a chemically defined medium was consistently improved to levels ∼400% compared to the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

Biochemistry Usa, 2007

The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)--tyrosine in the bios... more The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)--tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate L-tyrosine to generate (S)--tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the L-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

Biochemistry Usa, Jun 11, 2007

The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a... more The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPHdependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine (preQ 1 ), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a k cat ) 0.66 ( 0.04 min -1 , K M (preQ 0 ) ) 0.237 ( 0.045 µM, and K M (NADPH) ) 19.2 ( 1.1 µM. Based on sequence analysis and homology modeling we predicted previously that Cys 55 would be present in the active site and in proximity to the nitrile group of preQ 0 . Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ 0 was present. Furthermore, titrations of the enzyme with preQ 0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an R,unsaturated thioimide. Site-directed mutagenesis of Cys 55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ 0 . Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis.

Journal of Biological Chemistry

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified... more tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ 0 ) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ 0 as substrates, but not other 7-deazaguanine bases, and is able to modify tRNA from all three phylogenetic domains. The enzyme shows optimal activity at high temperature and acidic pH, consistent with the optimal growth conditions of M. jannaschii. The nature of the temperature dependence is consistent with a requirement for some degree of tRNA tertiary structure in order for recognition by the enzyme to occur.

Biochemistry, Jun 19, 2007

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the ... more The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

Organic letters, Jan 12, 2015

Bacteria strains and general DNA manipulation. Escherichia coli strains XL1 Blue (Stratagene) and... more Bacteria strains and general DNA manipulation. Escherichia coli strains XL1 Blue (Stratagene) and BL21(DE3) (EMD 4 Biosciences) were used for routine DNA cloning and overexpression, respectively. MTM producer S. argillaceus wild-type strain ATCC 12956, ΔmtmGII mutant strain M3G2 and ΔmtmGIV mutant strain M3G4 were stocked in this lab. Streptomyces lividans TK64 and S. argillaceus ΔmtmGIV mutant strain M3G4 were used as the host for protein expression in Streptomyces. Vectors pET28a(+) and pET30 Xa/LIC (Novagen) were used for protein expression in E. coli. Cultivation and DNA cloning in E. coli, and protoplast preparation and transformation of Streptomyces were carried out using standard protocols. PCR and constructions of protein expression plasmid of each gene were performed as follows: routine PCR was carried out using Pfu DNA polymerase (Stratagene) with denaturation at 96 °C for 3 min; followed by 25 cycles of 96 °C, 30 sec; 58 °C, 30 sec; 72 °C 2 min; with a final extension 72 °C for 5 min.

The Journal of biological chemistry, Jan 8, 2015

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discover... more A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent alpha-ketoglutarate:UMP dioxygenase and an L-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and L-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with...

Methods in enzymology, 2012

Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside ... more Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. T...

Methods in enzymology, 2009

Enediyne natural products are extremely potent antitumor antibiotics with a remarkable core struc... more Enediyne natural products are extremely potent antitumor antibiotics with a remarkable core structure consisting of two acetylenic groups conjugated to a double bond within either a 9- or 10-membered ring. Biosynthesis of this fascinating scaffold is catalyzed in part by an unusual iterative type I polyketide synthase, PKSE, that is shared among all enediyne biosynthetic pathways whose gene clusters have been sequenced to date. The PKSE is unusual in two main respects: (1) it contains an acyl carrier protein (ACP) domain with no sequence homology to any known proteins, and (2) it is self-phosphopantetheinylated by an integrated phosphopantetheinyl transferase (PPTase) domain. The unusual domain architecture and biochemistry of the PKSE hold promise both for the rapid identification of new enediyne natural products and for obtaining fundamental catalytic insights into enediyne biosynthesis. This chapter describes methods for rapid PCR-based classification of conserved enediyne biosyn...

Current opinion in drug discovery & development, 2008

Recent progress in the understanding of polyketide synthase (PKS) continues to fuel the growth of... more Recent progress in the understanding of polyketide synthase (PKS) continues to fuel the growth of combinatorial biosynthesis for natural product structural diversity. The structural analysis of many components of PKS, in particular for the modular type I 6-deoxyerythronilide B synthase (DEBS) involved in erythromycin biosynthesis, has provided structural imperatives for the observed biochemistry of DEBS and has enabled the generation of a working structural model of the entire DEBS system. New functions for PKS domains continue to be defined, such as the general control nonderepressible 5 (GCN5) N-acyltransferase strategy for polyketide chain initiation and the true identity of the elusive precursor for the methoxymalonylate extender unit. Novel molecular architectures have been continuously uncovered, including the 'AT-less' PKS and enediyne PKS, thereby expanding the known bacterial PKS paradigms beyond the prototypical type I, II and III PKSs. Finally, the genetic charact...

Biochemistry, Jan 19, 2007

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the ... more The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides i...

The Journal of biological chemistry, Jan 15, 2000

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified... more tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ(0)) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ(0) as substrates, but not other 7-deazaguan...

Journal of the American Chemical Society, Jan 24, 2005

The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. T... more The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. The biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety (boxed) of the C-1027 chromophore (1) from l-tyrosine (3) and its incorporation into 1 are catalyzed by six enzymes: SgcC, SgcC1, SgcC2, SgcC3, SgcC4, ShcC5. In vivo and in vitro characterization of these enzymes delineated this pathway, unveiling a novel strategy for beta-amino acid modification featuring beta-amino acyl-S-carrier protein intermediates. These findings shed new light into beta-amino acid biosynthesis and present a new opportunity to engineer the C-1027 biosynthetic machinery for the production of novel analogues as exemplified by 20-deschloro-C-1027 (4), 20-deschro-22-deshydroxy-C-1027 (5), and 22-deshydroxy-C-1027 (6).

Journal of the American Chemical Society, Jan 31, 2007

The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actin... more The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer Streptomyces carzinostaticus using the MDP enediyne PKS and associated genes. Furthermore, MDP was produced, and its apoprotein was isolated and N-terminal sequenced; the encoding gene, mdpA, was found to reside within the cluster. The biosynthesis of MDP is highlighted by two iterative type I PKSs--the enediyne PKS and a 6-methylsalicylic acid PKS; generation of (S)-3-(2-chloro-3-hydroxy-4-methoxyphenyl)-3-hydroxypropionic acid derived from L-alpha-tyrosine; a unique type of enediyne apoprotein; and a convergent biosynthetic approach to the final MDP chromophore. The results demonstrate a platform for engineering new enediynes by ...

Acta crystallographica. Section F, Structural biology and crystallization communications, 2005

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH... more QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 A. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ0. This crystal form diffracted to 2.25 A resolution.

Drug Discovery Today: Technologies, 2006

Combinatorial biosynthesis, the process of genetic manipulations of natural product biosynthetic ... more Combinatorial biosynthesis, the process of genetic manipulations of natural product biosynthetic machinery for structural diversity, depends on several factors, and discussed here are two critical factors: access to genetic information and biochemical characterization of enzymes. Examples of the former include using predictions for the biosynthesis of unusual chemical entities such as aminohydroxybenzoic acid starter units, methoxymalonylate extender units, the enediyne core and bacterial aromatic polyketides. The latter aspect includes the continued elucidation of domain functionalities of modular polyketide synthases and nonribosomal peptide synthases and novel biochemical pathways such as the biosynthesis of a cyclopropyl unit and a β-hydroxyl acid. Finally, examples of successful combinatorial biosynthesis for daptomycin and indolocarbozole compounds are discussed.:

Mol. BioSyst., 2015

Amide bond-containing (ABC) biomolecules are some of the most intriguing and functionally signifi... more Amide bond-containing (ABC) biomolecules are some of the most intriguing and functionally significant natural products with unmatched utility in medicine, agriculture and biotechnology. The enzymatic formation of an amide bond is therefore a particularly interesting platform for engineering the synthesis of structurally diverse natural and unnatural ABC molecules for applications in drug discovery and molecular design. As such, efforts to unravel the mechanisms involved in carboxylate activation and substrate selection has led to the characterization of a number of structurally and functionally distinct protein families involved in amide bond synthesis. Unlike ribosomal synthesis and thio-templated synthesis using nonribosomal peptide synthetases, which couple the hydrolysis of phosphoanhydride bond(s) of ATP and proceed via an acyl-adenylate intermediate, here we discuss two mechanistically alternative strategies: ATP-dependent enzymes that generate acylphosphate intermediates and ATP-independent transacylation strategies. Several examples highlighting the function and synthetic utility of these amide bond-forming strategies are provided.

ACS Symposium Series, 2007

Applied and Environmental Microbiology, 2015

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyce... more Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result many attempts have been made to develop ABM into a drug in the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfuramino acid (saa) metabolism that supplies a precursor for ABM biosynthesis could lead to an improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus, and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). Gene product AbmD appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplement in growth media increased ABM production by significantly changing relative concentrations of intracellular Cys and Hcy. A saa metabolic network of S. griseus was constructed. Pathways towards increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. ABM titer of one engineered strain SCAK3 in a chemically defined medium was consistently improved to levels ∼400% compared to the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

Biochemistry Usa, 2007

The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)--tyrosine in the bios... more The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)--tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate L-tyrosine to generate (S)--tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the L-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

Biochemistry Usa, Jun 11, 2007

The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a... more The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPHdependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine (preQ 1 ), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a k cat ) 0.66 ( 0.04 min -1 , K M (preQ 0 ) ) 0.237 ( 0.045 µM, and K M (NADPH) ) 19.2 ( 1.1 µM. Based on sequence analysis and homology modeling we predicted previously that Cys 55 would be present in the active site and in proximity to the nitrile group of preQ 0 . Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ 0 was present. Furthermore, titrations of the enzyme with preQ 0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an R,unsaturated thioimide. Site-directed mutagenesis of Cys 55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ 0 . Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis.

Journal of Biological Chemistry

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified... more tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ 0 ) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ 0 as substrates, but not other 7-deazaguanine bases, and is able to modify tRNA from all three phylogenetic domains. The enzyme shows optimal activity at high temperature and acidic pH, consistent with the optimal growth conditions of M. jannaschii. The nature of the temperature dependence is consistent with a requirement for some degree of tRNA tertiary structure in order for recognition by the enzyme to occur.

Biochemistry, Jun 19, 2007

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the ... more The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

Organic letters, Jan 12, 2015

Bacteria strains and general DNA manipulation. Escherichia coli strains XL1 Blue (Stratagene) and... more Bacteria strains and general DNA manipulation. Escherichia coli strains XL1 Blue (Stratagene) and BL21(DE3) (EMD 4 Biosciences) were used for routine DNA cloning and overexpression, respectively. MTM producer S. argillaceus wild-type strain ATCC 12956, ΔmtmGII mutant strain M3G2 and ΔmtmGIV mutant strain M3G4 were stocked in this lab. Streptomyces lividans TK64 and S. argillaceus ΔmtmGIV mutant strain M3G4 were used as the host for protein expression in Streptomyces. Vectors pET28a(+) and pET30 Xa/LIC (Novagen) were used for protein expression in E. coli. Cultivation and DNA cloning in E. coli, and protoplast preparation and transformation of Streptomyces were carried out using standard protocols. PCR and constructions of protein expression plasmid of each gene were performed as follows: routine PCR was carried out using Pfu DNA polymerase (Stratagene) with denaturation at 96 °C for 3 min; followed by 25 cycles of 96 °C, 30 sec; 58 °C, 30 sec; 72 °C 2 min; with a final extension 72 °C for 5 min.

The Journal of biological chemistry, Jan 8, 2015

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discover... more A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent alpha-ketoglutarate:UMP dioxygenase and an L-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and L-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with...

Methods in enzymology, 2012

Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside ... more Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. T...

Methods in enzymology, 2009

Enediyne natural products are extremely potent antitumor antibiotics with a remarkable core struc... more Enediyne natural products are extremely potent antitumor antibiotics with a remarkable core structure consisting of two acetylenic groups conjugated to a double bond within either a 9- or 10-membered ring. Biosynthesis of this fascinating scaffold is catalyzed in part by an unusual iterative type I polyketide synthase, PKSE, that is shared among all enediyne biosynthetic pathways whose gene clusters have been sequenced to date. The PKSE is unusual in two main respects: (1) it contains an acyl carrier protein (ACP) domain with no sequence homology to any known proteins, and (2) it is self-phosphopantetheinylated by an integrated phosphopantetheinyl transferase (PPTase) domain. The unusual domain architecture and biochemistry of the PKSE hold promise both for the rapid identification of new enediyne natural products and for obtaining fundamental catalytic insights into enediyne biosynthesis. This chapter describes methods for rapid PCR-based classification of conserved enediyne biosyn...

Current opinion in drug discovery & development, 2008

Recent progress in the understanding of polyketide synthase (PKS) continues to fuel the growth of... more Recent progress in the understanding of polyketide synthase (PKS) continues to fuel the growth of combinatorial biosynthesis for natural product structural diversity. The structural analysis of many components of PKS, in particular for the modular type I 6-deoxyerythronilide B synthase (DEBS) involved in erythromycin biosynthesis, has provided structural imperatives for the observed biochemistry of DEBS and has enabled the generation of a working structural model of the entire DEBS system. New functions for PKS domains continue to be defined, such as the general control nonderepressible 5 (GCN5) N-acyltransferase strategy for polyketide chain initiation and the true identity of the elusive precursor for the methoxymalonylate extender unit. Novel molecular architectures have been continuously uncovered, including the 'AT-less' PKS and enediyne PKS, thereby expanding the known bacterial PKS paradigms beyond the prototypical type I, II and III PKSs. Finally, the genetic charact...

Biochemistry, Jan 19, 2007

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the ... more The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides i...

The Journal of biological chemistry, Jan 15, 2000

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified... more tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ(0)) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ(0) as substrates, but not other 7-deazaguan...

Journal of the American Chemical Society, Jan 24, 2005

The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. T... more The enediyne antitumor antibiotic C-1027 chromoprotein is produced by Streptomyces globisporus. The biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety (boxed) of the C-1027 chromophore (1) from l-tyrosine (3) and its incorporation into 1 are catalyzed by six enzymes: SgcC, SgcC1, SgcC2, SgcC3, SgcC4, ShcC5. In vivo and in vitro characterization of these enzymes delineated this pathway, unveiling a novel strategy for beta-amino acid modification featuring beta-amino acyl-S-carrier protein intermediates. These findings shed new light into beta-amino acid biosynthesis and present a new opportunity to engineer the C-1027 biosynthetic machinery for the production of novel analogues as exemplified by 20-deschloro-C-1027 (4), 20-deschro-22-deshydroxy-C-1027 (5), and 22-deshydroxy-C-1027 (6).

Journal of the American Chemical Society, Jan 31, 2007

The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actin... more The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer Streptomyces carzinostaticus using the MDP enediyne PKS and associated genes. Furthermore, MDP was produced, and its apoprotein was isolated and N-terminal sequenced; the encoding gene, mdpA, was found to reside within the cluster. The biosynthesis of MDP is highlighted by two iterative type I PKSs--the enediyne PKS and a 6-methylsalicylic acid PKS; generation of (S)-3-(2-chloro-3-hydroxy-4-methoxyphenyl)-3-hydroxypropionic acid derived from L-alpha-tyrosine; a unique type of enediyne apoprotein; and a convergent biosynthetic approach to the final MDP chromophore. The results demonstrate a platform for engineering new enediynes by ...

Acta crystallographica. Section F, Structural biology and crystallization communications, 2005

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH... more QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 A. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ0. This crystal form diffracted to 2.25 A resolution.

Drug Discovery Today: Technologies, 2006

Combinatorial biosynthesis, the process of genetic manipulations of natural product biosynthetic ... more Combinatorial biosynthesis, the process of genetic manipulations of natural product biosynthetic machinery for structural diversity, depends on several factors, and discussed here are two critical factors: access to genetic information and biochemical characterization of enzymes. Examples of the former include using predictions for the biosynthesis of unusual chemical entities such as aminohydroxybenzoic acid starter units, methoxymalonylate extender units, the enediyne core and bacterial aromatic polyketides. The latter aspect includes the continued elucidation of domain functionalities of modular polyketide synthases and nonribosomal peptide synthases and novel biochemical pathways such as the biosynthesis of a cyclopropyl unit and a β-hydroxyl acid. Finally, examples of successful combinatorial biosynthesis for daptomycin and indolocarbozole compounds are discussed.:

Mol. BioSyst., 2015

Amide bond-containing (ABC) biomolecules are some of the most intriguing and functionally signifi... more Amide bond-containing (ABC) biomolecules are some of the most intriguing and functionally significant natural products with unmatched utility in medicine, agriculture and biotechnology. The enzymatic formation of an amide bond is therefore a particularly interesting platform for engineering the synthesis of structurally diverse natural and unnatural ABC molecules for applications in drug discovery and molecular design. As such, efforts to unravel the mechanisms involved in carboxylate activation and substrate selection has led to the characterization of a number of structurally and functionally distinct protein families involved in amide bond synthesis. Unlike ribosomal synthesis and thio-templated synthesis using nonribosomal peptide synthetases, which couple the hydrolysis of phosphoanhydride bond(s) of ATP and proceed via an acyl-adenylate intermediate, here we discuss two mechanistically alternative strategies: ATP-dependent enzymes that generate acylphosphate intermediates and ATP-independent transacylation strategies. Several examples highlighting the function and synthetic utility of these amide bond-forming strategies are provided.

ACS Symposium Series, 2007