Widespread promiscuous alkaline phosphatases underscore early microbial phosphite utilization

Widespread promiscuous alkaline phosphatases underscore early microbial phosphite utilization | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Widespread promiscuous alkaline phosphatases underscore early microbial phosphite utilization View ORCID Profile Morito Sakuma , View ORCID Profile Naoki Konno , Sevan Gholipour , View ORCID Profile John Z Chen , View ORCID Profile Nobuhiko Tokuriki doi: https://doi.org/10.1101/2025.04.29.651336 Morito Sakuma 1 Michael Smith Laboratories, University of British Columbia , Vancouver, British Columbia V6T 1Z4, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Morito Sakuma Naoki Konno 2 Department of Biological Sciences, Graduate School of Science, The University of Tokyo , 7-3- 1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 3 Universal Biology Institute, Graduate School of Science, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Naoki Konno Sevan Gholipour 1 Michael Smith Laboratories, University of British Columbia , Vancouver, British Columbia V6T 1Z4, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site John Z Chen 4 Research School of Chemistry, Australian National University , Canberra, Australia 5 ARC Centre of Excellence in Synthetic Biology, Research School of Biology, Australian National University , Canberra, ACT 2601, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for John Z Chen Nobuhiko Tokuriki 1 Michael Smith Laboratories, University of British Columbia , Vancouver, British Columbia V6T 1Z4, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nobuhiko Tokuriki For correspondence: tokuriki{at}msl.ubc.ca Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Phosphate is often a limiting resource, directly affecting the availability of key biomolecules such as nucleotides. To cope with phosphate scarcity, bacteria have evolved enzymes that utilize alternative phosphorus compounds, including phosphite (Pt). Although a few enzymes oxidize Pt to produce phosphate, the enzymes responsible for Pt oxidation in many environmental bacteria remain unidentified, and the role of microbial Pt oxidation in the global phosphorus cycle is not yet fully understood. In this study, we performed bioinformatic analyses of three Pt-oxidizing enzymes: the native Pt oxidase phosphite dehydrogenase (PtxD), and two promiscuous Pt oxidases, alkaline phosphatase (AP) and carbon-phosphorus (CP) lyase. Among these, AP was found to be widely distributed across bacteria since the early stages of their evolution. In contrast, PtxD emerged later in a limited number of bacterial lineages that had lost AP. Our biochemical characterizations revealed that most extant and reconstructed ancestral APs tested exhibited Pt oxidation activity. Moreover, disruption of active-site residues diminished Pt oxidase activity in AP, while only partially affecting its native function. This promiscuous function of AP reveals an overlooked mechanism in bacterial phosphate metabolism and underscores the role of Pt in the cycling of bioavailable phosphorus in ecosystems. Introduction Phosphate is essential for life, forming key components such as ATP, nucleic acids, and cell membranes. Nevertheless, phosphate is often limited in many ecosystems, directly constraining the growth of organisms [ 1 - 3 ]. To overcome this limitation, microbes utilize alternative sources of dissolved organic phosphorus (DOP) via enzymes such as phosphatases [ 3 - 7 ]. Phosphonates are also abundant and serve as a source of phosphate through the cleavage of carbon-phosphorus (C-P) bonds by CP lyases and phosphonatases [ 1 - 3 , 8 , 9 ]. In addition to these organic sources, phosphite (Pt), a reduced and highly soluble inorganic phosphorus compound, is of particular interest due to its ecological role in the global phosphorus cycle and microbial metabolism [ 1 - 3 , 10 ]. Pt is thought to have been prevalent in early Earth ecosystems, especially under reducing environments before the Great Oxidation Event (GOE) (> 2.5 Ga) [ 1 , 3 , 11 - 13 ]. Its abundance subsequently declined after the GOE (< 2.3 Ga), due to a reduction in Pt-generating processes and shifts in Earth’s redox conditions. However, Pt is still frequently detected up to micromolar concentrations across diverse modern environments, including soil, aquatic systems, and geothermal settings [ 1 - 3 , 14 ], highlighting its ongoing ecological relevance. Pt is a kinetically stable compound, requiring an activation energy of 370 kJ to break its P–H bond [ 11 ]. Therefore, microbes rely on specialized enzymes—such as Pt oxidases—to convert Pt into phosphate. To date, however, only one enzyme family, phosphite dehydrogenase (PtxD), has been classified as catalyzing Pt oxidation as its primary function [ 15 , 16 ]. PtxD efficiently catalyzes Pt oxidation ( k cat / K M ∼ 10 5 M -1 s -1 ) using NAD + as a cofactor and has been identified as a part of the ptx operon ( Fig. 1A ) [ 3 , 17 ]. In addition to generating phosphate from Pt to support bacterial growth, PtxD is also capable of utilizing phosphite as an exclusive electron donor, facilitating the coupling of Pt oxidation to the reduction of carbon dioxide, sulfate, or nitrate in the process known as dissimilatory phosphite oxidation (DPO) [ 3 , 18 ]. Despite its efficiency, PtxD is surprisingly rare in bacterial genomes, primarily identified in species such as Pseudomonas stutzeri ( P. stutzeri ) and certain anaerobes [ 3 , 19 , 20 ]. A recent phylogenetic study by Boden et al . also suggested that ptxD (encoding PtxD) genes underwent significant expansion only after the GOE [ 21 ]. These observations raise the question of whether PtxD is the sole enzyme responsible for Pt oxidation in nature, and suggest that global microbially driven Pt oxidation may depend on only a limited number of bacterial species harboring PtxD. However, studies have shown that a substantial proportion (10–67%) of bacterial isolates can grow in media containing Pt as the sole phosphorus source, suggesting that other, yet unidentified, enzymes may also be involved in Pt oxidation [ 22 ]. Download figure Open in new tab Fig. 1. Distribution of phosphite (Pt) oxidase genes on the phylogenetic tree of bacterial genomes. (A) Phosphate production and transportation in bacteria. Alkaline phosphatase (AP, PDB: 1ALK), located in the periplasm, catalyzes the hydrolysis of phosphate monoester substrates and the Pt oxidation reaction to produce phosphate. PafA (PDB: 5TJ3), another member of the AP superfamily, also catalyzes the hydrolysis of phosphate monoesters. The phosphate-binding protein PstS (PDB: 1A40) and pho regulon proteins transport phosphate into the cytoplasm. PtxD (PDB: 4E5K) catalyzes the Pt oxidation reaction in the cytoplasm using NAD + as a cofactor. Carbon-phosphorus (CP) lyase (PDB: 4XB6) degrades phosphonate monoester substrates to produce phosphate and catalyzes Pt oxidation. Native and promiscuous functions are indicated by thick and thin arrows, respectively. (B) Distribution of the phoA (red), pafA (green), ptxD (blue), and phnJ (cyan) genes mapped onto the bacterial phylogenetic tree. The presence of each gene is indicated by lines on the circle. Bacterial phyla are also mapped onto the tree. The positions of the twelve phoA genes exhibiting Pt oxidation activity are shown, along with the phoA genes in bacterial genomes that have the highest sequence identity percentage (SIP) to them. SIP was calculated using BLASTp. (C) The abundance of enzyme genes across all bacterial phyla. (D), (E) Metagenomic analysis of the phoA and ptxD genes. (D) Violin plots showing the relative frequencies of the recA, phoA , and ptxD genes across all environments. The recA gene was used as a control [ 30 ]. *** indicates a p-value < 0.001 in the Kolmogorov-Smirnov (KS) test. (E) Relative frequencies of the recA, phoA , and ptxD genes in each environment. N . D . indicates that no ptxD genes were detected in animal-associated environments. Two other enzymes, CP lyase (encoded by the phn genes) and alkaline phosphatase (encoded by the phoA gene), have been reported to exhibit Pt oxidation activity as a non-primary or promiscuous function. While CP lyase and its phn operon in Escherichia coli ( E. coli ) and P. stutzeri facilitate phosphate assimilation from phosphonate substrates [ 1 , 8 ], they can also contribute to Pt oxidation ( Fig. 1A ) [ 1 , 23 ]. However, the phn genes also show limited distribution among environmental microbes and are believed to have expanded after the GOE [ 21 ]. AP is a widely distributed periplasmic phosphomonoesterase (PMEase) in bacteria that produces phosphate from phosphoester compounds [ 3 , 4 ] ( Fig. 1A ). APs often catalyze multiple promiscuous reactions, including Pt oxidation [ 24 – 27 ]. Interestingly, although both AP and CP lyase exhibit significantly lower Pt oxidation activity than PtxD—with AP showing a turnover rate nearly 10,000-fold lower—it has been demonstrated that heterologous expression of E. coli AP and P. stutzeri CP lyase enables bacterial growth in Pt-containing media [ 24 , 28 ]. These observations raise a pivotal question: Do these promiscuous enzymes play a physiological role in microbial Pt oxidation, or are they merely serendipitous, non-physiological promiscuous activities? A comprehensive understanding of the distributions of PtxD and promiscuous Pt oxidases among microbes is essential for elucidating the role of microbial Pt oxidation in global phosphorus cycling. In this study, we address these questions through comprehensive bioinformatic analyses and biochemical characterizations. We found that the ptxD gene is exceedingly rare, present in only a few specific bacterial taxa, whereas the phoA gene was the most abundant and widely distributed among diverse bacterial phyla. Through experimental characterization of twelve APs, including reconstructed ancestral APs, we demonstrated that Pt oxidase activity is far more widespread among bacterial APs than previously recognized. We propose that PtxD, a specialized Pt oxidase, likely evolved later to confer a growth advantage in Pt-rich environments. In contrast, multifunctional APs may represent the most ancient Pt oxidase, playing a significant role in Pt oxidation from the pre-GOE to the modern era. Finally, we discuss the potential contribution of phosphatase-mediated Pt oxidation to global phosphorus cycling. Results The distribution of the ptxD, phnJ, pafA , and phoA genes among bacteria We first curated and classified homologous sequences for three enzyme families known to catalyze Pt oxidation ( Fig. S1 and see Methods )—PtxD (encoded by the ptxD gene), CP lyase ( phnJ gene, which encodes the catalytic subunit (PhnJ) of the CP lyase complex), and AP ( phoA gene ) — as well as PafA ( pafA gene), an efficient PMEase homologous to AP. We analyzed their distribution across a previously constructed bacterial phylogenetic tree comprising 32,225 representative bacterial species (one genome per species) across 105 phyla [ 29 ] ( Fig. 1B ). While the ptxD gene is the only native Pt oxidase gene, its orthologs were exceedingly rare, found in only 1.2% of bacterial genomes (373 genomes), mostly within Proteobacteria and to a lesser extent in Actinobacteria and Cyanobacteria ( Fig. 1B-C ). While the phnJ gene was more abundant (10%, 3,346 genes) than the ptxD gene, it was primarily distributed in Proteobacteria ( Fig. 1B-C ). In contrast, the phoA gene was widely distributed, identified in approximately 30% of the bacterial genome (9,588 genomes) across various phyla ( Fig. 1B-C ). The broad distribution of the phoA gene is especially notable compared to the pafA gene, which was found in only 16% of bacterial genomes (5,277 genes) and was predominantly distributed in Bacteroidota (69% of the pafA genes, 3,626 genes) ( Fig. 1B-C ). Overall, the phoA / pafA gene pair was present in about 40% of bacterial genomes ( Fig. 1B-C ). To investigate the distribution of the ptxD and phoA genes among environmental microbes, we searched for their homologs in a large set of metagenomic data [ 30 ]. We compared the abundance of the ptxD and phoA genes with that of the recA gene, a common single-copy bacterial marker gene. Both genes exhibited similar frequency to the genomic data; the phoA gene was found in about 30% of the recA gene, and the ptxD gene appeared only in less than 5% ( Fig. 1D ). Moreover, both genes are found in diverse environments; however, the ptxD gene was not observed in animal-associated environments ( Fig. 1E ). We also examined the distribution of the phosphate-binding protein PstS ( pstS gene) ( Fig. 1A ), which shares the same regulon as the phoA gene [ 31 ] and is considered to have existed since the Archean Eon [ 21 ]. Indeed, the pstS gene was prevalent among bacterial genomes (22,463 genes, 70% of total genomes) ( Fig. 1B-C ). Therefore, the phoA gene, which has also been inferred to be part of the gene repertoire of the last universal common ancestor (LUCA) [ 32 ], may have been present in bacteria since ancient times, possibly as early as the Archean Eon, as a member of the same pho regulon. We further analyzed the co-occurrence and mutual exclusivity of the ptxD, phoA , and phnJ genes in the phylogenetic tree. Interestingly, while the phoA and phnJ genes appeared to be partially co-distributed, the phoA and ptxD genes exhibited a complementary distribution pattern ( Fig. 1B ). Co-occurrence analysis using evolCCM [ 33 ] revealed no significant co-occurrence between the phoA and phnJ genes (p < 0.005), whereas the phoA and ptxD genes showed a significantly negative co-occurrence (p < 0.001) ( Fig. S2 ), suggesting possible functional redundancy or conflict between the genes. Notably, the pafA gene also exhibited significant negative co-occurrence with the ptxD gene (p < 0.001), suggesting that the ptxD gene may have a function inversely related to phosphatase-associated activity ( Fig. S2 ). To further explore the patterns of gene gain, loss, and coexistence during the evolutionary process, we used PastML, which predicts the presence or absence of genes at each ancestral node in the phylogenetic tree [ 29 , 34 ] ( Fig. S3 ). While the phoA and phnJ genes exhibited co-occurrence in their ancestral states, the phoA and ptxD genes showed minimal overlap in their ancestral distribution ( Fig. S3 ). Interestingly, the gain of the ptxD gene was largely restricted to lineages in which the phoA gene had been lost ( Fig. S3A ), suggesting that the ptxD gene may have provided a similar function to the phoA gene. Sequence diversity of APs As the phoA genes were the most widely distributed among phosphite (Pt) oxidases, we next explored the diversity and phylogenetic distribution within the AP superfamily ( Fig. 2A ). We collected sequences belonging to the AP superfamily (total of 32,843 sequences), which includes various enzyme families such as phosphodiesterases, sulfatases, PafA, and mutases. Using Sequence similarity networks (SSNs), we isolated a sequence cluster that contained APs (15,722 sequences) ( Fig. S1A ), identified by conserved active site structures including the bimetallic binding site, catalytic residues, and nucleophilic serine, as well as annotated AP sequences ( Fig. S4–6 ). Within the AP family, we identified six major subclusters (AP1–6), among which AP3 and AP4 were further subdivided into four (AP3-1 to AP3-4) and two (AP4-1 and AP4-2) subgroups, respectively ( Fig. 2A ). We also generated a phylogenetic tree of the AP family using representative sequences from each subcluster (total of 3,091 sequences) ( Fig. 2B ). The phyla of the host organisms were mapped onto both the SSNs and the phylogenetic tree, color-coded by phylum ( Fig. 2A-B ). Many characterized bacterial and fungal APs, including Bacillus subtilis ( bs AP), Escherichia coli ( ec AP), Thermotoga maritima ( tm AP), and Saccharomyces cerevisiae ( sc AP), were found in AP3 ( Fig. 2A and Fig. S6 ). All characterized mammalian APs belonged to AP4, while the cold-active Vibrio sp . AP ( vs AP) was located in AP6 [ 35 ]. Download figure Open in new tab Fig. 2. Divergence of AP sequences. (A) SSN analysis of six AP subclusters (AP1-6) identified in the clustering of the AP superfamily database ( Fig. S1A ). The E-value cut-offs for AP1, 2, 5, 6, AP3, and AP4 were 1 × 10 −100 , 1 × 10 −40 , and 1 × 10 −125 , respectively. The ten most abundant phyla are represented by nodes in different colors. AP3 and AP4 are further divided into subgroups (AP3-1, 2, 3, 4 and AP4-1, 2). Pie charts show the phylum distribution within each AP subcluster and subgroup. (B) Phylogenetic tree analysis of AP subcluster. The distribution of the AP subclusters and their associated phyla is mapped onto the phylogenetic tree. Enzymes used for expression and activity measurements, ancestral enzymes, and annotated enzymes are indicated in red, orange, and green, respectively. Interestingly, the distribution of phyla across the AP subcluster was quite diverse. Especially, Pseudomonadota, Bacteroidota , and Fungi appeared in multiple AP subclusters: Pseudomonadota in AP1, AP3, AP4, and AP6; Fungi in AP1 and AP3-2; and Bacteroidota in AP2, AP3, and AP5. In some cases, the phylum distribution was resolved at the subgroup level, e . g ., AP4-1 and AP4-2; Metazoa / Pseudomonadota , AP3-2; Bacillota/Pseudomonadota/Fungi , AP3-4; Actinomycetota / Pseudomonadota . Among 9,588 bacterial genomes in the GTDB database that possess the phoA gene, 21% (2,030 genomes) contained multiple copies of phoA ( Fig. S7A ), which were clustered in certain bacterial classes such as Gammaproteobacteria (in Pseudomonadota ), and Bacteroidia ( in Bacteroidota ) ( Table S1 ). We also observed that the multiple copies of the phoA gene within these classes were assigned to different AP subclusters ( Fig. S7B ): AP2/AP5, AP3/AP5, AP5/AP5 were commonly found in Bacteroidota ; AP3/AP4, AP3/AP6, AP3/AP3, and AP4/AP4 were seen in Pseudomonadota ( Table S1 ). These observations are consistent with our earlier findings that extensive gene gain and loss of the phoA gene have occurred among bacteria during their evolutionary divergence ( Fig. S3 ). Experimental validation of Pt oxidation activity in bacterial AP A previous study by Yang et. al . demonstrated that only E. coli alkaline phosphatase ( ec AP) catalyzed the Pt oxidation reaction, while other APs, including those from B. subtilis , calf, and shrimp, did not [ 24 ]. However, it remains unclear how prevalent Pt oxidase activity is among other bacterial APs. To address this, we synthesized genes and experimentally characterized thirteen bacterial APs across diverse subgroups ( Fig. S6 ). Among these, only seven APs exhibited soluble fractions in SDS-PAGE and measurable phosphomonoesterase (PMEase) activity in lysate assays ( Fig. S8 and Fig. S9 ). Of these seven APs, five belong to AP3 ( Pseudomonadota : ec AP, Acinetobacter gerneri : ag AP, Halieaceae bacterium : hb AP, unclassified Rheinheimera : rs AP, and Thermotoga maritima : tm AP), one belongs to AP6 ( Pseudomonadota : unclassified Vibrio : vs AP), and one belongs to AP2 ( Bacteroidota : Elizabethkingia meningoseptica : em AP) ( Fig. 2 ). While crystal [ 35 , 36 ] and predicted structures of these APs revealed several insertion/deletion sites, they all maintained the AP fold with the canonical bimetallic binding site, catalytic residues, and nucleophile serine ( Fig. 3A ). We purified these enzymes and measured both PME and Pt oxidation activities. All the APs exhibited efficient PME activity and, intriguingly, also demonstrated Pt oxidation activity comparable to that of ec AP, which provides a growth advantage to the E. coli Shuffle strain (Δ ptxD , Δ phoA ) in Pt media ( Fig. 3B-C and Fig. S10 ). Download figure Open in new tab Fig. 3. Bulk ensemble kinetic assay of phosphomonoesterase (PMEase) and phosphite (Pt) oxidation functions of homologous APs. (A) Structure of extant and ancestral APs. The ec AP (PDB: 1ALK) and vs AP (PDB: 3E2D) have crystal structures solved in previous studies. Structures of other APs were estimated using AlphaFold 2. Domains with deletions or insertions are shown in red, blue, and green, respectively. (B), (C) PMEase and Pt oxidation activities of extant and ancestral APs (mean ± SD , n = 3). Asterisks indicate enzymes for which activity could not be measured. We further analyzed the functional profile of inferred ancestral AP enzymes (ancAPs). Using ancestral sequence reconstruction with GRASP [ 37 ], we predicted the sequences of common ancestors for several subclusters and subgroups: ancAP2, ancAP3-1, ancAP3-2, ancAP3-3, ancAP3-4, and ancAP4 ( Fig. 2 , Fig. 3A , and Fig. S11 ). All ancAPs, except ancAP4, were expressed in the soluble fraction in E. coli ( Fig. S8 and Fig. S9 ). We found that three out of five of the ancAPs—ancAP3-2, ancAP3-3, and ancAP3-4—showed Pt oxidation activity, while all soluble ancAPs exhibited phosphomonoesterase (PMEase) activity ( Fig. 3B-C ). These ancAPs were located at early branching points in the phylogenetic tree ( Fig. 2B ), suggesting that Pt oxidation activity might have been present in the early stages of AP evolution. This function appears to have been inherited in certain lineages and is likely still common among extant APs, although other lineages have lost their ability to oxidize Pt. However, since Pt oxidation by APs is significantly weaker than PMEase activity ( Fig. 3B-C ), it is unlikely to be the primary function of the AP family. Finally, we analyzed the divergence of Pt oxidation functions among archaeal APs, which are evolutionarily distant from bacterial APs, and PafA, another class of PMEase enzymes belonging to the phosphodiesterase family. We identified 203 phoA genes from a total of 4,416 representative archaeal species ( Fig. S12A ), with more than 80% of the sequences containing the canonical bimetallo binding site and nucleophile serine ( Fig. S12B-D ). Compared to bacterial phoA genes, archaeal phoA genes showed a more limited distribution. Additionally, only one, two, and zero ptxD, pafA , and phnJ genes, respectively, were identified in archaeal genomes. These findings suggest that archaea may employ different enzymes for PME and Pt oxidation, or, as reported in previous studies, may not utilize Pt as a phosphorus source [ 21 ]. We selected two archaeal APs, Pyrococcus abyssi [ 38 ] and Halobacterium salinarum [ 39 ], and tested their catalytic activities ( Fig. S12B, E, F ). While H. salinarum AP was insoluble in bacterial expression systems, we found that P. abyssi AP was soluble ( Fig. S8C ) and exhibited PME activity (0.94 ± 0.07, ((nM/s)/nM enzyme), mean ± SD , n = 3, at 60°C), consistent with previous reports [ 38 ]. However, we did not observe Pt oxidation activity with purified P. abyssi AP, suggesting that Pt oxidation may not be widespread among archaeal APs. In the case of PafA, we expressed and purified PafA homologs from Elizabethkingia meningoseptica ( em PafA) and Flavobacterium johnsoniae ( fj PafA), with em PafA sharing identical metal-binding residues and the same primary function as ec AP [ 26 ]. While fj PafA was insoluble, em PafA was expressed in a soluble form ( Fig. S8D ) and exhibited PME activity (315 ± 6 ((nM/s)/nM), mean ± SD , n = 3), similar to that of AP. Interestingly, em PafA also showed Pt oxidation activity (1.1 ± 0.24 ((nM/h)/nM enzyme, mean ± SD , n = 3), suggesting that other enzyme classes with native PME activity may also possess Pt oxidation activity. Key active site residues of APs are optimized for Pt oxidation The Pt oxidation activity by extant and ancestral APs suggests that there may have been selection pressure leading to the retention of this function in the APs. To investigate this further, we analyzed the diversity of active site residues within the AP family. In ec AP, six amino acid residues—101, 102, 166, 153, 322, and 328—have been shown to play key roles in PME and promiscuous functions ( Fig. 4A ) [ 26 , 27 ]. A multiple sequence alignment of the active site residues in each AP subcluster revealed that all APs share the same active site structure, while an overall multiple sequence alignment of APs showed interesting differences between subgroups. While positions 101 (Asp), 102 (Ser), 166 (Arg), and 322 (Glu) are highly conserved across all AP sequences (91-100% conservation), diversities were observed at positions 153 and 328. Position 153 varies between Asp (26%) and His (70%), and position 328 diversifies between His (32%), Lys (14%), and Trp (35%) ( Fig. 4A ). Moreover, AP sequences exhibited specific 153/328 combinations, with three combinations— D153/K328, H153/W328, and H153/H328—found in more than 80% of AP sequences ( Fig. 4B ). Notably, we observed that preference for specific combinations is evident at both the domain and phylum levels, with phyla predominantly possessing one or two specific combinations ( Fig. 4C and Fig. S13 ). Since the 153/328 combination is conserved at the phylum level, this pattern was also reflected in each subcluster of the SSN, where a specific 153/328 combination is well-preserved ( Fig. 4D and Fig. S14 ). Download figure Open in new tab Fig. 4. Effect of the 153/328 combination on AP functions. (A) Alignment of the active site structures. Extant and ancestral enzymes shown in Fig. 3A were used for the alignment. Dark and green spheres represent zinc and magnesium ions identified by the crystal structure of ec AP (PDB: 1ALK). (B) Ratio of the specific 153/328 combination in the AP subclusters. Each graph shows the percentage of the amino acid residue at position 153 when the residue at position 328 is H, K, or W, respectively. (C) Sequence count of the 153/328 combination in the most abundant seven phyla. (D) Ratio of the specific 153/328 combination in each AP subcluster. The color code is the same as in Fig. 4C . (E), (F) PMEase and Pt oxidation activities of swapping mutants (mean ± SD , n = 3). Activities of the mutants were normalized to those of their wild-type enzymes. Asterisks indicate enzymes for which activity could hardly be measured. Is the conservation of the 153/328 combination a consequence of functional constraints? In other words, are specific combinations in each subcluster necessary for the function of APs? To analyze the mutational effects of the 153/328 combinations, we generated a series of swapping mutants based on the six APs and measured their PMEase and Pt oxidation activities ( Fig. 4E-F ). The wild-type sequences of em AP, rp AP, and vs AP contain H153/W328, while those of ec AP, ag AP, and hb AP have D153/K328. We selected the most dominant swapping mutations—H153/H328, H153/W328, and D153/K328. Swapping combinations strongly affected PMEase activity in various ways depending on the genetic background. For example, the swapping mutations negatively affected the PMEase activities of ec AP, vs AP, and hb AP, while they positively influenced em AP. rp AP exhibited both positive and negative effects depending on the mutations ( Fig. 4E ). Mutations at positions 153 and 328 resulted in various effects, including changes in the rate-limiting step, altered magnesium affinity, and shifts in phosphate positioning [ 27 , 40 ]. The complex interplay of these effects, along with interactions with different surrounding residues in homologous enzymes, leads to enzyme-specific variations in catalytic properties. In contrast, swapping mutations consistently showed negative effects on Pt oxidation activity across all tested APs ( Fig. 4F ). For ec AP, ag AP, and rp AP, Pt oxidation decreased by more than fivefold. Moreover, Pt oxidation could not be detected in the mutated hb AP and em AP, even though these mutations had a positive impact on the PMEase activity of em AP. Thus, the wild-type combination in each enzyme appears to be somewhat subjected to selection pressure and optimized for Pt oxidation, but it is not essential for PMEase activity. Since the balance of activities was easily disrupted by mutations in the active site, organisms may frequently use AP that already retain an optimized functional spectrum for organism growth, as reflected by their frequent transfer ( Fig. 2 ). Discussion This study highlights the overlooked ecological significance of the promiscuous Pt oxidation function and its potential contribution to the global phosphorus cycle. While microbes have evolved to utilize diverse phosphorus compounds, early life likely first relied on the most readily available forms—primarily inorganic phosphates—to expand their metabolic capabilities. However, the limited solubility of phosphate and the intense competition for its assimilation may have driven the evolution of diverse enzymatic pathways to process various DOP sources [ 1 - 3 ]. Given that nucleic acids and numerous phosphorylated metabolites are indispensable for organism growth [ 41 , 42 ], phosphatases would have emerged in early life to recycle and produce bioavailable phosphate. Indeed, a previous study suggested that inferred ancestral metabolic pathways in LUCA contained several phosphatases, including AP [ 32 ]. Our observations suggest that these phosphatases may have provided ancient microbes with the ability to access other phosphorus sources, i . e ., Pt, via their multifunctionality [ 25 - 27 , 42 ]. Despite the low levels of Pt oxidase activity observed in APs, they were sufficient levels to support E. coli growth in Pt-containing media ( Fig. S10 ) [ 24 ], suggesting potential physiological roles in natural environments, particularly where microbes have access to a mixture of diverse phosphorus sources, including inorganic phosphate, DOPs, and Pt [ 1 - 3 , 5 , 7 ]. A more efficient and specialized Pt oxidase, PtxD, appeared later than AP and likely spread among bacteria that require much higher catalytic Pt oxidase activity [ 1 , 18 - 20 ]. Pt concentration gradually declined as Earth’s atmosphere oxidized after the GOE, with reduced inputs, such as meteorite impacts and lightning events, contributing to this decrease [ 1 , 3 , 12 , 43 , 44 ]. However, Pt continues to be produced abiotically [ 3 , 45 - 48 ] and remains ubiquitous, including in anoxic environments [ 18 - 20 , 49 ]. Thus, it is highly likely that accessing phosphite as a phosphorus source can give an advantage to diverse modern bacteria. In this study, we found that 47% of bacterial genomes (15,305 out of 32,255) harbor at least one gene encoding a potential Pt oxidase, including the phoA, pafA, ptxD , or phnJ genes ( Fig. 1B ). Additionally, given the structural similarity of their active sites [ 25 , 26 ], it is also possible that other phosphatases can catalyze Pt oxidation, which may explain previous observations that 10–67% of bacterial isolates from environmental samples are capable of growing in Pt-containing media [ 22 ]. Further characterization of promiscuous Pt oxidation by phosphatases and CP lyases, along with investigation of the distribution of environmental microbes encoding these genes, is expected to provide new insights into the role of Pt in bacterial phosphorus metabolism. Author contributions M.S. conceived the project, designed experiments, performed experiments, analyzed results, and wrote the manuscript; N.K., S.G., and J.Z.C. performed experiments, analyzed results, and wrote the manuscript; N.T. conceived the project, designed experiments and wrote the manuscript. Competing interest declaration The authors declare no competing financial interest. 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Share Widespread promiscuous alkaline phosphatases underscore early microbial phosphite utilization Morito Sakuma , Naoki Konno , Sevan Gholipour , John Z Chen , Nobuhiko Tokuriki bioRxiv 2025.04.29.651336; doi: https://doi.org/10.1101/2025.04.29.651336 Share This Article: Copy Citation Tools Widespread promiscuous alkaline phosphatases underscore early microbial phosphite utilization Morito Sakuma , Naoki Konno , Sevan Gholipour , John Z Chen , Nobuhiko Tokuriki bioRxiv 2025.04.29.651336; doi: https://doi.org/10.1101/2025.04.29.651336 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40364) Molecular Biology (17163) Neuroscience (88537) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)