News and Events
Nicole Leal's Publications
Hachimoji DNA and RNA: A genetic system with eight building blocks
Hoshika H, Leal N, Kim MJ, Kim MS, Karalkar NB, Kim HJ, Bates AM, Watkins Jr. NE, SantaLucia HA, Meyer AJ, DasGupta S, Piccirilli JA, Ellington AD, SantaLucia Jr. J, Georgiadis MM, Benner SA
(2019) 22 Feb 2019: Vol. 363, Issue 6429, pp. 884-887. DOI: 10.1126/science.aat0971
We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.
"Skinny" and "Fat" DNA: Two New Double Helices
Hoshika S, Singh I, Switzer C, Molt RW Jr, Leal NA, Kim MJ, Kim MS, Kim HJ, Georgiadis MM, Benner SA
J. Am. Chem. Soc.
(2018) Sep 19;140(37):11655-11660. doi: 10.1021/jacs.8b05042. Epub 2018 Sep 10
According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.
Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System
Karalkar NB, Leal NA, Kim MS, Bradley KM, Benner SA
ACS Synthetic Biology
, American Chemical Society (2016) doi: 10.1021/acssynbio.5b00276
As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ~92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ~86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.
Transcription, Reverse Transcription, and Analysis of RNA Containing Artificial Genetic Components
Nicole A. Leal, Hyo-Joong Kim, Shuichi Hoshika, Myong-Jung Kim, Matthew A. Carrigan, and Steven A. Benner
ACS Synthetic Biology
, American Chemical Society (2015) Apr 17;4(4):407-13. doi: 10.1021/sb500268n
Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.
Ribonucleosides for an Artificially Expanded Genetic Information
Hyo-Joong Kim, Nicole A. Leal, Shuichi Hoshika, Steven A. Benner
J. Org. Chem.
(2014) 79 (7), pp 3194-3199
Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2?-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson?Crick complement,imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this "second-generation" AEGIS Z:P pair to add amino acids encoded by mRNA.
Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns
Laos R, Shaw R, Leal NA, Gaucher E, Benner S.
(2013) 52, 5288-5294
Artificial genetic systems have been developed
by synthetic biologists over the past two decades to include
additional nucleotides that form additional nucleobase pairs
independent of the standard T:A and C:G pairs. Their use in
various tools to detect and analyze DNA and RNA requires
polymerases that synthesize duplex DNA containing unnatural
base pairs. This is especially true for nested polymerase chain
reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are
used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA
polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard
nucleotides, 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with
6-amino-5-nitro-3-(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed
evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template.
Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed
"heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change
in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the
template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the
general rule in directed evolution that "you get what you select for".
Conversion strategy using an expanded genetic alphabet to assay nucleic acids
Yang, Z., Durante, M., Glushakova, L., Sharma, N., Leal, N., Bradley, K., Chen, F., Benner, S. A.
Methods to detect DNA and RNA (collectively
xNA) are easily plagued by noise, false positives, and false
negatives, especially with increasing levels of multiplexing in
complex assay mixtures. Here, we describe assay architectures
that mitigate these problems by converting standard xNA
analyte sequences into sequences that incorporate nonstandard
nucleotides (Z and P). Z and P are extra DNA building blocks
that form tight nonstandard base pairs without cross-binding
to natural oligonucleotides containing G, A, C, and T
(GACT). The resulting improvements are assessed in an
assay that inverts the standard Luminex xTAG architecture,
placing a biotin on a primer (rather than on a triphosphate).
This primer is extended on the target to create a standard
GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a
polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that
is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces
higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures
improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of
competition from any natural oligonucleotides, even in complex biological samples.
Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups
Hutter, D; Kim, MJ; Karalkar, N; Leal, NA; Chen, F; Guggenheim, E; Visalakshi, V; Olejnik, J; Gordon, S; Benner, SA
Nuc. Nuc. Nuc. acids
29 (11) , Taylor & Francis Group 879-895 (2010)
Nucleoside triphosphates having a 3'-ONH(2) blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH(2) group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH(2) blocking group in "next generation sequencing."
2'-Deoxy-1-methylpseudocytidine, a stable analog of 2'-deoxy-5-methylisocytidine
Kim, HJ; Leal, NA; Benner, SA
Bioorg. Med. Chem.
17 (10) 3728-3732 (2009)
2 '-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2'-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2 '-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2 '-deoxycytidine and 2 '-deoxyguanosine pair. (C) 2009 Elsevier Ltd. All rights reserved.
Synthetic Biology for Improved Personalized Medicine
Benner, SA; Hoshika, S; Sukeda, M; Hutter, D; Leal, NA; Yang, ZY; Chen, F
Nucleic Acids Symp. Ser.
52 (1) 243-244 (2008) doi: 10.1093/nass/nrn123
Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than $10,000 (as opposed to $100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and the Westheimer Institute have developed several of these, which will be the topic of this talk.
Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis
Ruhland, A; Leal, N; Kima, PE
9 (1) 84-96 (2007)
Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NF kappa B and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NF kappa B signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.
PduL is an evolutionarily distinct phosphotransacylase involved in B-12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2
Liu, Y; Leal, NA; Sampson, EM; Johnson, CLV; Havemann, GD; Bobik, TA
189 (5) 1589-1596 (2007)
Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B-12-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V-max was 51.7 +/- 7.6 mu mol min(-1) mg(-1), and the K-m values for propionyl-PO42- and acetyl-PO42- were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.
In vivo expression of human ATP : cob(I)atamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8
Erger, KE; Conlon, TJ; Leal, NA; Zori, R; Bobik, TA; Flotte, TR
J. Gene Med.
9 (6) 462-469 (2007)
Background Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B-12, adenosylcobalamin (AdoCbl). AdoCbl is. a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Methods The human ATR cDNA was cloned into a recombinant adenoassociated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/B16 mice at a dose of 1 x 10(10) and 1 x 10(11), particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Results Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken P-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. Conclusions These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments. Copyright (C) 2007 John Wiley & Sons, Ltd.
Dynamic assembly of primers on nucleic acid templates
Leal, NA; Sukeda, M; Benner, SA
Nucl. Acids Res.
34 4702-4710 (2006)
A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of 1 µM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.
Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase
Leal, NA; Olteanu, H; Banerjee, R; Bobik, TA
J. Biol. Chem.
279 (46) 47536-47542 (2004)
The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.
PduP is a Coenzyme-A-Acylating Propionaldehyde Dehydrogenase Associated with the Polyhedral Bodies Involved in B<sub>12</sub>-dependent 1,2-propanediol Degradation by <i>Salmonella enterica</i> serovar Typhimurium LT2
Leal, NA; Havemann, GD; Bobik, TA
180 (5) 353-361 (2003)
Salmonella enterica forms polyhedral bodies involved in coenzyme-B12-dependent 1,2-propanediol degradation. Prior studies showed that these bodies consist of a proteinaceous shell partly composed of the PduA protein, coenzyme-B12-dependent diol dehydratase, and additional unidentified proteins. In this report, we show that the PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica. A PCR-based method was used to construct a precise nonpolar deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal medium and expressed 105-fold less propionaldehyde dehydrogenase activity (0.011 micromol min(-1) mg(-1)) than did wild-type S. enterica grown under similar conditions (1.15 micromol min(-1) mg(-1)). An Escherichia coli strain was constructed for high-level production of His8-PduP, which was purified by nickel-affinity chromatography and shown to have 15.2 micromol min(-1) mg(-1) propionaldehyde dehydrogenase activity. Analysis of assay mixtures by reverse-phase HPLC and mass spectrometry established that propionyl-CoA was the product of the PduP reaction. For subcellular localization, purified His8-PduP was used as antigen for the preparation of polyclonal antiserum. The antiserum obtained was shown to have high specificity for the PduP protein and was used in immunogold electron microscopy studies, which indicated that PduP was associated with the polyhedral bodies involved in 1,2-propanediol degradation. Further evidence for the localization of the PduP enzyme was obtained by showing that propionaldehyde dehydrogenase activity co-purified with the polyhedral bodies. The fact that both Ado-B12-dependent diol dehydratase and propionaldehyde dehydrogenase are associated with the polyhedral bodies is consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. enterica on 1,2-propanediol.
Identification of the Human and Bovine ATP:cob(I)alamin Adenosyltransferase cDNAs Based on Complementation of a Bacterial Mutant
Leal, NA; Park, SD; Kima, PE; Bobik, TA
J. Biol. Chem.
278 (11) 9227-9234 (2003)
In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in the diagnosis and treatment of this serious disease.
Functional genomic, biochemical and genetic characterization of the <i>Salmonella pduO</i> gene, an ATP:cob(I)alamin adenosyltransferase gene
Johnson, CLV; Pechonick, EM; Park, SD; Havemann, GD; Leal, NA; Bobik, TA
183 1577-1584 (2001)
Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.