Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440 Jose´I. Jime´nez*†, Angeles´ Canales‡, Jesu´s Jime´nez-Barbero‡, Krzysztof Ginalski§, Leszek Rychlewski¶, Jose´L. Garcı´a*, and Eduardo Dı´az*ʈ Departments of *Molecular Microbiology and ‡Protein Science, Centro de Investigaciones Biolo´gicas–Consejo Superior de Investigaciones Cientı´ficas,28040 Madrid, Spain; §Interdisciplinary Centre for Mathematical and Computational Modeling, University of Warsaw, 02-106 Warsaw, Poland; and ¶BioInfoBank Institute, 60-744 Poznan, Poland Edited by David T. Gibson, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, and approved May 21, 2008 (received for review March 11, 2008) The aerobic catabolism of nicotinic acid (NA) is considered a model microorganisms for Ͼ50 years (1, 2, 5–7), the complete set of system for degradation of N-heterocyclic aromatic compounds, genes encoding this pathway as well as the structural–functional some of which are major environmental pollutants; however, the relationships of most of the enzymes involved in this process have complete set of genes as well as the structural–functional relation- not been reported and analyzed so far in any organism. In many ships of most of the enzymes involved in this process are still bacteria, the aerobic degradation of NA produces 2,5DHP as a unknown. We have characterized a gene cluster (nic genes) from central intermediate, which is further degraded according to a Pseudomonas putida KT2440 responsible for the aerobic NA deg- scheme (maleamate pathway) that has been outlined (Fig. 1) (1, radation in this bacterium and when expressed in heterologous 2, 6, 7). In this work, we show that Pseudomonas putida KT2440, hosts. The biochemistry of the NA degradation through the for- a well studied and paradigmatic bacterium renowned for its mation of 2,5-dihydroxypyridine and maleamic acid has been ability to metabolize a wide range of aromatic compounds (8, 9), revisited, and some gene products become the prototype of new is able to use NA aerobically as a sole carbon, nitrogen, and types of enzymes with unprecedented molecular architectures. energy source. We have been able to identify and characterize in Thus, the initial hydroxylation of NA is catalyzed by a two- this bacterium a gene cluster (nic genes) responsible for the component hydroxylase (NicAB) that constitutes the first member aerobic NA degradation. The biochemistry of aerobic NA of the xanthine dehydrogenase family whose electron transport degradation through the maleamate pathway has been revisited, chain to molecular oxygen includes a cytochrome c domain. The and we have demonstrated that some gene products of this Fe2؉-dependent dioxygenase (NicX) converts 2,5-dihydroxypyri- pathway become prototypes of new types of enzymes with dine into N-formylmaleamic acid, and it becomes the founding unprecedented molecular architectures. Some ecological and member of a new family of extradiol ring-cleavage dioxygenases. evolutionary considerations regarding the nic genes are also MICROBIOLOGY Further conversion of N-formylmaleamic acid to formic and mal- discussed. eamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some Results and Discussion members of the ␣/␤-hydrolase fold superfamily. This work allows The nic Genes Are Responsible for NA Catabolism in P. putida KT2440. exploration of the existence of orthologous gene clusters in sap- The in silico analysis of the P. putida KT2440 genome revealed rophytic bacteria and some pathogens, where they might stimulate a gene cluster, named nicTPFEDCXRAB (Fig. 1), some of whose studies on their role in virulence, and it provides a framework to gene products showed significant similarities to proteins involved develop new biotechnological processes for detoxification/bio- in the catabolism of N-heterocyclic aromatic compounds [sup- transformation of N-heterocyclic aromatic compounds. porting information (SI) Table S1]. When P. putida KT2440 was tested with different N-heterocycles (pyridoxal, pyridoxamine, ring-cleavage dioxygenase ͉ nicotinic acid hydroxylase ͉ pyridine, 2- and 3-hydroxypyridine, NA, isonicotinic acid, pico- heterocyclic compounds linic acid, quinoline, and isoquinoline) as sole carbon and energy sources, we observed growth only in NA (Fig. 1C), thus impli- icotinic acid (NA) is a carboxylic derivative of pyridine that cating the nic cluster in NA catabolism. To confirm this assump- Nis widely distributed in nature as part of pyridine cofactors tion, several P. putida KT2440 knockout mutants were con- (e.g., NAD and NADP) and alkaloids (e.g., nicotine and structed (Table S2). Disruption of the nicA, nicB, nicC, nicD, and anabasine), and it is essential (vitamin B3) for those organisms nicX genes did not allow the corresponding P. putida KT2440 C that are not able to carry out its synthesis (1). NA is also a carbon mutant strains to grow in NA as the sole carbon source (Fig. 1 ). The nic cluster also contained two genes, nicP and nicT, encod- and nitrogen source for different bacteria and some fungi (1, 2), ing a potential porin and permease transport system, respec- and the biochemical pathways involved in the degradation of this N-heterocyclic aromatic compound have been used as a source of novel and unusual enzyme activities as well as metabolic Author contributions: J.I.J., J.L.G., and E.D. designed research; J.I.J. performed research; intermediates, e.g., 6-hydroxynicotinic acid (6HNA) and 2,5- A.C., J.J.-B., K.G., and L.R. contributed new reagents/analytic tools; J.I.J., J.J.-B., K.G., L.R., dihydroxypyridine (2,5DHP), that can funnel the degradation of J.L.G., and E.D. analyzed data; and J.I.J. and E.D. wrote the paper. toxic compounds of major environmental concern, such as The authors declare no conflict of interest. nicotine and hydroxypyridines (1, 2), and are of pharmacological This article is a PNAS Direct Submission. and agrochemical value (3). So far, the only fully elucidated NA †Present address: Department of Microbial Biotechnology, Centro Nacional de Biotecno- degradation pathway is the anaerobic route from Eubacterium logı´a–ConsejoSuperior de Investigaciones Cientı´ficas,28049 Madrid, Spain. barkeri (4). ʈTo whom correspondence should be addressed. E-mail: [email protected]. Although the aerobic NA degradation has been considered as This article contains supporting information online at www.pnas.org/cgi/content/full/ a model system for the degradation of N-heterocyclic aromatic 0802273105/DCSupplemental. compounds and its biochemistry has been studied in different © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802273105 PNAS ͉ August 12, 2008 ͉ vol. 105 ͉ no. 32 ͉ 11329–11334 Downloaded by guest on October 2, 2021 A CO OH HCOOH CH O Nic X O N formic ac id H COOH NicAB COOH Nic C OH NF M Nic D COOH Nic F CO OH Nic E COOH N HO N HO N CONH2 + CO OH HO OC NH 4 NA 6HNA 2,5 DHP maleami c ac id maleic acid fuma ric acid B C nicP nicT nicF nicE nicD nicC nicX nicR nicA nicB Ppu Bor 60 46 71 61 78 64 55 43 62 Bcen 49 58 64 78 58 55 43 49 56 57 Reut 57 41 65 62 82 64 54 44 58 62 Ctest 52 40 70 63 78 64 51 45 58 Aaven 53 39 70 62 78 66 51 45 58 Dacid 53 40 70 63 77 65 51 45 57 Bxen 36 80 61 53 43 60 71 Fig. 1. Aerobic NA degradation via the maleamate pathway. (A) Scheme of the aerobic NA degradation pathway in P. putida KT2440. NicAB, NA hydroxylase (green); NicC, 6HNA monooxygenase (blue); NicX, 2,5DHP dioxygenase (red); NicD, NFM deformylase (yellow); NicF, maleamate amidohydrolase (orange); NicE, maleate isomerase (pink). The hatched arrow shows the previously proposed 2,5DHP ring-cleavage reaction. (B) Genetic organization of the nic cluster in different bacteria. Genes are functionally annotated following the color code indicated in A. Regulatory genes encoding NicR-type and TetR-type regulators are indicated by black and dotted arrows, respectively. Solid, dotted, and striped green arrows indicate the nicB2 gene/domain, the nicB1 gene/domain, and the nicA gene, respectively. Porin and MFS transport genes are indicated by hatching arrows. Genes encoding subunits of putative ABC transporters are represented by vertically striped arrows. Numbers within the arrows indicate the percent amino acid sequence identity with the ortholog gene product from P. putida KT2440. The abbreviations and accession codes for the different gene clusters are detailed in SI Text.(C) Growth of P. putida KT2440 (portion 1), Pseudomonas sp. MT14 (pNIC) (portion 2), P. putida KT2440dnicB (portion 3), and Pseudomonas sp. MT14 (pBBR1MCS-5) (portion 4) in MC minimal medium containing 5 mM NA as the sole carbon source. tively, and a regulatory gene (nicR) (Fig. 1 and Table S1; see also droxylase that converts NA into 6HNA, constituting the first SI Text). aerobic NA hydroxylase whose primary structure is known. Growth in NA could be restored when the P. putida knockout Hydroxylases of N-heterocyclic aromatic compounds incor- mutants were complemented with a broad-host-range plasmid porate oxygen derived from a water molecule into the product, (pNIC) harboring a 14-kb DNA cassette containing the com- and they typically contain a redox center, formed by a molyb- plete nic cluster (Table S2). Interestingly, the pNIC plasmid also denum ion coordinated to an organic cofactor (molybdopterin conferred the ability to use NA as sole carbon source to other cytosine dinucleotide, MCD), two [2Fe-2S] clusters, and usually bacteria, such as Pseudomonas sp. MT14 (Table S2), that are FAD (Fig. 2B), which transports electrons from the reducing unable to degrade this aromatic compound (Fig.