The Enzymatic Conversion of Phosphonates to Phosphate by Bacteria
Total Page:16
File Type:pdf, Size:1020Kb
Available online at www.sciencedirect.com The enzymatic conversion of phosphonates to phosphate by bacteria 1 Siddhesh S Kamat and Frank M Raushel Phosphonates are ubiquitous organophosphorus compounds annually into the environment in the form of herbicides that contain a characteristic C–P bond which is chemically inert and detergent wastes. With such large quantities of and hydrolytically stable. Bacteria have evolved pathways to phosphonates being released into the environment, there metabolize these phosphonate compounds and utilize the is a significant interest in understanding the mechanisms products of these pathways as nutrient sources. This review by which phosphonates are degraded or metabolized by aims to present all of the known bacterial enzymes capable of bacterial species [1]. The abundance and universal preva- transforming phosphonates to phosphates. There are three lence of phosphonates in the environment has led to the major classes of enzymes known to date performing such evolution of several bacterial species that are able to transformations: phosphonatases, the C-P lyase complex and metabolize and utilize phosphonates as carbon and phos- an oxidative pathway for C–P bond cleavage. A brief phorus sources [2–4]. There are three known classes of description of each class is presented. enzymes or enzymatic systems that have been mechan- istically characterized which are capable of breaking the Addresses C–P bonds of phosphonate compounds. These include Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, TX 77843, United States phosphonate hydrolases, the C-P lyase complex, and an oxidative pathway. Corresponding author: Raushel, Frank M ([email protected]) 1 Present address: The Skaggs Institute for Chemical Biology and Phosphonate hydrolases Department of Chemical Physiology, The Scripps Research Institute, La Phosphonate hydrolases have been generically referred to Jolla, CA 92037, United States. as ‘phosphonatases’. The characteristic feature of the substrates for the phosphonatases is the presence of an electron withdrawing b-carbonyl group that facilitates Current Opinion in Chemical Biology 2013, 17:589–596 bond delocalization and allows the heterolytic cleavage This review comes from a themed issue on Mechanisms of the C–P bond. The phosphonate substrates hydrolyzed Edited by Hung-wen Liu and Tadhg Begley by this group of enzymes include phosphonopyruvate For a complete overview see the Issue and the Editorial (PnPy), phosphonoacetate (PAA) and phosphonoacetal- dehyde (Pald) (Scheme 1). Proteins belonging to the Available online 2nd July 2013 phosphonatase class of enzymes have evolved from differ- 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights ent enzyme superfamilies, and have been characterized reserved. mechanistically and structurally. http://dx.doi.org/10.1016/j.cbpa.2013.06.006 The first reported phosphonopyruvate hydrolase (PPH) was identified from cell free extracts of an environmental Phosphonates are organophosphorus compounds that isolate capable of utilizing phosphonoalanine as a carbon, contain a characteristic carbon–phosphorus (C–P) bond. nitrogen and phosphorus source, from Burkholderia cepacia This bond is chemically inert, hydrolytically stable and Pal6 [5]. The PPH reaction is presented in Figure 1a, resistant to photolysis. Phosphonates are prevalent in all where phosphonopyruvate is converted to pyruvate and primitive life forms, where they can exist as integral orthophosphate. On the basis of amino acid sequence components of membrane phosphonolipids, such as 2- identity, the gene for PPH has also been identified in aminoethylphosphonic acid and 2-amino-3-phosphono- Variovorax sp. Pal2, another environmental sample propionic acid (Scheme 1). The presence of phosphonates obtained from a soil isolate [6]. PPH belongs to the in these lipid membranes confers rigidity to the mem- phospho(enol)pyruvate (PEP) mutase/isocitrate lyase branes and protects the organisms against light and superfamily of enzymes [7 ]. PPH has a 40% amino acid degradation from phosphatases [1–3]. Biologically sequence identity to PEP mutase and has a (b/a)8-barrel relevant exopolysaccharides and glycoproteins also con- structural fold. The monomers associate as a tetramer and tain phosphonate moieties. Phosphonates are found the 8th a-helix is swapped between two dimers [7 ]. extensively in antibiotics such as fosfomycin and phos- There are three available structures for PPH from Var- phonothrixin, the herbicide glyphosate and the industrial iovorax sp. Pal2: apo-enzyme (PDB id: 2HRW), PPH 2+ detergent additive amino-trimethylene phosphonate complexed with Mg and phosphonopyruvate (PDB 2+ (Scheme 1) [1–3]. It is estimated that in the US alone id: 2HJP), and PPH complexed with Mg and oxalate 2+ more than 20 000 tons of phosphonates are released (PDB id: 2DUA). The active site Mg anchors the www.sciencedirect.com Current Opinion in Chemical Biology 2013, 17:589–596 590 Mechanisms Scheme 1 O O O– O O – O H2N P O R1 O P N P O H O R2 H2N R1 O – O – O R2 2-aminoethylphosphonate glyphosate lipid 2-amino-3-phosphono- propionic acid lipid O O OH O O – O – P O O P N –O P –O P –O O O O – O – HO –O P O– fosfomycin phosphonothrixin O amino-trimethylene phosphonate O O O O O O O – – P O –O P O– H P O – O O– O– O– phosphonopyruvate phosphonoacetate phosphonoaetaldehyde Current Opinion in Chemical Biology Structures of important phosphonates. phosphonopyruvate substrate. On the basis of the avail- The structure of PAH from S. meliloti has been deter- able structures of PPH and mechanistic experiments, a mined for the apo-enzyme (PDB id: 3SZY), complexed consensus catalytic mechanism analogous to that of PEP with phosphonoacetate (PDB id: 3T02), acetate (PDB id: mutase has been proposed (Figure 2) [5,6,7 ,8]. PPH does 3SZZ), vanadate (PDB id: 3T00) and phosphonoformate not utilize any other phosphonate compounds besides (PDB id: 3T01). On the basis of these crystal structures, phosphonopyruvate [6]. PAH possess a catalytic core of the alkaline phosphatase superfamily and uses a conserved threonine residue to Phosphonoacetate is biogenically available to bacteria form the phospho-enzyme adduct in the catalytic cycle. from the degradation of 2-aminoethylphosphonate However, this enzyme possesses a unique capping (2AEP) [9 ]. The first phosphonoacetate hydrolase domain analogous to the nucleoside pyrophosphatase/ (PAH) activity was found in Pseudomonas fluorescens phosphodiesterase enzyme family that is critical for 23F, a bacterial isolate from the sludge of a laundry waste shielding the substrate from solvent during turnover. A treatment plant in Ireland [10]. PAH catalyzes the detailed reaction mechanism for the PAH has been pro- hydrolysis of phosphonoacetate to yield acetate and posed (Figure 3) [9 ,13 ]. orthophosphate (Figure 1b). PAH belongs to the alkaline phosphatase superfamily [11]. Members of this super- Phosphonoacetaldehyde (Pald) is formed biologically by family have an active site consisting of a binuclear metal the action of 2AEP-pyruvate transaminase, which uses center with a highly conserved serine or threonine residue 2AEP and pyruvate as substrates and yields Pald and L- that is utilized to form a covalent phospho-enzyme adduct alanine as products. Pald is subsequently hydrolyzed by as an integral part of the catalytic mechanism [12]. There phosphonoacetaldehyde hydrolase (PaldH) to form acet- are two PAH superfamily enzymes that have been exten- aldehyde and orthophosphate (Figure 1c). PaldH sively characterized: PAH from P. fluorescens 23F [9 ] and belongs to the haloalkanoic acid dehydrogenase Sinorhizobium meliloti 1021 (phnA) [13 ]. Both of these (HAD) superfamily of enzymes [14,15]. Members of enzymes have high specificity for zinc, but the PAH from the HAD superfamily perform diverse set of transform- 2+ 2+ 2+ S. meliloti can be activated with Mn or Fe . The ations that require the Mg dependent formation of a structure of PAH from P. fluorescens has been determined covalent intermediate to an active site aspartate residue in a complex with phosphonoformate (PDB id: 1EI6). [16]. PaldH from Bacillus cereus is the most extensively Current Opinion in Chemical Biology 2013, 17:589–596 www.sciencedirect.com Phosphonate metabolism Kamat and Raushel 591 Figure 1 substrate to activate Pald. The histidine residue activates a water molecule for hydrolytic attack and the aspartate (a) residue forms the phospho-enzyme complex for cleavage O – O– O O O of the C–P bond. A comprehensive mechanism has been PPH O proposed for PaldH based on the mechanistic character- P O + HO P O ization and the available X-ray structures (Figure 4) –O O– O– O [17,18,19]. (b) CP lyase complex O O– O O– The C-P lyase complex has been studied for over four PAH decades. The C-P lyase activity is very promiscuous and –O P O + HO P O –O was first studied in cell free extracts of several bacterial – O O strains capable of metabolizing a broad spectrum of phosphonates including unactivated substrates such as (c) methylphosphonate (MPn) and ethylphosphonate (EtPn) O – – O 2-AEP-pyruvate O [20,21]. The C-P lyase activity in bacteria is governed by aminotr ansferase the 14-cistron phnCDEFGHIJKLMNOP operon. It is upre- H2N P O H P O gulated under conditions of phosphate starvation, and is O– O– regulated by the PhoR/PhoB two component signaling O system [22,23]. The C-P lyase activity was predicted to O– O O– PaldH arise from the gene products of the phn operon forming a H P O + HO P O membrane-localized multienzyme complex in bacteria H O– O [22,23]. The gene products PhnC, PhnD and PhnE are Current Opinion in Chemical Biology phosphonate transporters, based on sequence analysis and phenotypes obtained from transposon-based mutants Reactions catalyzed by phosphonate hydrolases. These include grown on different phosphonates [22,23]. The proteins reactions catalyzed by phosphonopyruvate hydrolase (PPH), PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL and PhnM are phosphonoacetate hydrolase (PAH) and phosphonoacetaldehyde absolutely critical for C-P lyase activity and form the hydrolase (PaldH).