Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis

Abstract Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase). The C. parapsilosis GBase (CpGBase) ORF was PCR amplified, cloned, and sequenced. Further, the functional CpGBase protein expressed in Saccharomyces cerevisiae functioned as GBase and 3-guanidinopropionase (GPase). S. cerevisiae cannot grow on GB or GP. However, the transformants expressing CpGBase acquired the ability to utilize and grow on both GB and GP. The expressed CpGBase protein was enriched and analyzed for substrate saturation and product inhibition by γ-aminobutyric acid and β-alanine. In contrast to the well-characterized AnGBase, CpGBase from C. parapsilosis is a novel ureohydrolase and showed hyperbolic saturation for GB and GP with comparable efficiency (Vmax/KM values of 3.4 and 2.0, respectively). With the paucity of structural information and limited active site data available on ureohydrolases, CpGBase offers an excellent paradigm to explore this class of enzymes.


Introduction
Ur eohydr olase superfamil y (E.C. 3.5.3.x)consists of hydr ol ytic enzymes like arginase (E.C. 3.5.3.1),agmatinase, formiminoglutamase , procla vaminate amino hydrolase (PAH), 4-guanidinobutyrase (GBase; E.C. 3.5.3.7), and 3guanidinopr opionase (GP ase;E.C. 3.5.3.17).T hese enzymes clea ve off ur ea fr om their r espectiv e guanidinium gr oup-containing substrates (with the exception of PAH) and they ar e v ery specific in recognizing their substrate side chains (Kumar et al. 2015 ).GBase catalyses the hydrolysis of 4-guanidinobutyrate (GB) and produces γ -aminobutyric acid (GABA) and urea.The GABA enters the TCA cycle by its conversion into succinate while the ur ea formed hydr ol yzed to NH 3 for use as the nitrogen source.Wher eas, GP ase catal yzes the hydr ol ysis of 3-guanidinopr opionic acid (GP) to urea and β-alanine .T he β-alanine is then metabolized via malonic semialdehyde and acetyl CoA to enter the TCA cycle; ho w e v er, this pathway is not operational in all organisms.GBase from a few organisms is a broadly specific enzyme and can also act on GP.The metabolism of GP assumes significance as it is used as a dietary supplement in sports medicine (Oudman et al. 2013 ) and is being investigated as a potential antihypertensive drug (Karamat et al. 2015 ).Ho w ever, there is only one report so far on a specific GPase in P. aeruginosa PAO1 (Lee et al. 2011 ).
In the ascomycetes fungus Aspergillus niger , two ur eohydr olases namel y, ar ginase and GBase were functionally annotated and fun-gal growth on different guanidium compounds was also characterized (Dave et al. 2012, Kumar et al. 2015 ).Inter estingl y, GB is efficiently utilized by A. niger , but GP is not.The A. niger GBase acts 25 times more efficiently on GB than on GP, and the gbu (GBase knoc k out) str ain was unable to gr ow on GP (Sar a gadam et al. 2019 ).This suggested the absence of a specific GPase in this fungus.But A. niger can effectiv el y utilize both urea and β-alanine, the two pr oducts of GP hydr ol ysis .And the GBase o v er expr essing A. niger strain shows better growth on GP.These observations collectively point to the poor GPase activity of A. niger GBase (AnGBase).
Our molecular understanding of ur eohydr olase substr ate specificity is still evolving (Dutta et al. 2019, Sekula 2020, Maturana et al. 2021, Funck et al. 2022 ).Not many GBase structur es ar e av ailable (Nakada and Itoh 2002, 2005, Lee et al. 2011 ), and hence rational substrate specificity engineering through sitedir ected m uta genesis is difficult.At first, we attempted to impr ov e AnGBase specificity to w ar ds GP through EMS mutagenesis and screening of A. niger conidia, albeit with little success (Sar a gadam et al. 2019 ).A dir ected e volution a ppr oac h (using err or-pr one PCR) was also undertaken; for this, in vitro , mutated AnGBase ORFs were transformed into Saccharomyces cerevisiae 12T7cI ( car1::kanMX4, ura3 ).The inability of S. cerevisiae to utilize GB, GP, and arginine (the car1 background) served well to set up a GPase screen.While screening for such transformants (around 1.0 lakh colonies; T. Sar a gadam, personal comm unication), a yeast contaminant that gr e w efficientl y on GP was isolated.This ne w yeast isolate, a Candida parapsilosis strain, was e v aluated for its GBase and GPase profiles .T he characterization of a novel broad specificity GBase/GPase from this yeast is presented here.

Organisms and growth conditions
Aspergillus niger NCIM 565 (and its transformants) was maintained on potato dextrose agar slants .T he yeast strains were maintained on yeast nitrogen base (YNB) agar supplemented with 5 mM ammonium sulfate (AS) and 2% glucose.Sacc harom yces cerevisiae 12T7cI ( car1::kanMX4, ura3 ), an ar ginase-negativ e yeast m utant, was used to screen error-prone PCR products of A. niger GBase cDNA (Sar a gadam et al. 2019 ).Synthetic minimal medium [glucose 2.0 g, yeast nitrogen base (YNB and AS in 1:3 ratio) 0.66 g, distilled water 100 ml, agar 2.0 g] was used for this screening and the transformants were later replica plated on synthetic minimal medium supplemented with either 5 mM GB or GP instead of AS.The same medium without AS was used to grow S. cerevisiae 12T7cI transformants and the C. parapsilosis strain by supplementing an equimolar nitr ogen source, namel y, AS (7.5 mM), GB (5 mM), GP (5 mM), AR (3.75 mM), GABA (15 mM), β-alanine (15 mM), or urea (7.5 mM).Similar media without agar were prepared for liquid growth studies.

Molecular techniques to manipulate DNA
QIAGEN DNeasy Plant Mini kit was used to pr epar e genomic DNA from yeast and A. niger for genomic PCR.Restriction digestions and ligations were performed essentially according to standard pr ocedur es (Sambr ook and Russell 2001 ).The restriction enzymes and T4 DNA ligase were from New England Biolabs or MBI Fermentas.Esc heric hia coli str ain XL1 Blue was used in all the transformation experiments for cloning purposes.Competent E. coli XL1 Blue cells were prepared by PEG method (Nishimura et al. 1990 ) and stored in glycerol/PEG at -80 • C. Competent E. coli BL21 (DE3) and E. coli Rosetta gamiB (DE3) pLysS cells were prepared by CaCl 2 method (Sambrook and Russell 2001 ) and stored at −80 • C until further use.
When transformed with the desired plasmid, E. coli XL1-Blue, E. coli BL21 (DE3), and E. coli Rosetta gamiB were grown and maintained on Luria Bertani broth (containing 100 μg/ml ampicillin).Yeast transformation was performed by lithium acetate method (Ito et al. 1983 ).For this, the S. cerevisiae 12T7cI strain was inoculated into 10 mL YPD (Yeast extract 5 g/l, bactopeptone 10 g/l, and dextrose 20 g/l) medium.The plasmids bearing GBase expression constructs were linearized with Sca I and used to transform A. niger NCIM 565 protoplasts (Kumar et al. 2015 ).The tr ansformants wer e single-spored and characterized for GBase expression.

Identification of the yeast isolate as C. parapsilosis
The yeast isolate (e v entuall y identified as C. parapsilosis ) was grown in synthetic minimal medium supplemented with 5 mM AS, for genomic DNA isolation.The gDNA was isolated from a 24-h grown culture and was subjected to PCR amplification using ITS1 (5 -tccgtaggtgaacctgcgg-3 ) and ITS4 (5 -tcctccgcttattgatatgc-3 ) primers .T he amplified PCR product was purified and sequenced using ITS1.The evolutionary history was inferred using the Maximum Parsimony (MP) method.The most parsimonious tree (length = 931) was generated with the consistency index of 0.631325, the retention index of 0.763524, the composite in-dex of 0.512570 for all sites and the parsimon y-informativ e sites (0.482032).The MP tree was obtained using the Subtree-Pruning-Regrafting algorithm with search level 1, in which the initial trees were obtained by the random addition of sequences (10 replicates).T he analysis in volved 24 nucleotide sequences.Codon positions included were 1st + 2nd + 3rd + Noncoding.There was a total of 586 positions in the final dataset.Evolutionary analyses were conducted in MEGA6.The yeast isolate was thereby identified as C. parapsilosis .

Identification and cloning of putati v e GBase ORF from C. parapsilosis
The GBase and/or GPase loci are not functionally annotated in the C. parapsilosis genome .T he A. niger GBase (AnGBase) protein sequence (AHL44994) served to search for putative GBase and/or GPase sequences in the Candida Genome Database (CGD).Based on the identity scores, forw ar d (CparGPF; 5ccttgtc catatg aa gttgcttccacttttaa gc-3 ) and r e v erse (CparGPR; 5gac ctcgag tcagttggcacccttgtaaacttg-3 ) primers were designed to PCR amplify the putative GBase ORF from C. parapsilosis gDNA.The Nde I and Xho I fr a gment of this amplicon was cloned in the pET.Nat.Ar g v ector by r eplacing A. niger ar ginase ( Figur e S1, Supporting Information ).The resultant plasmid (pET23a-CparGB) was transformed in E. coli XL1 blue; the ampicillin-resistant transformants wer e scr eened and c har acterized by colon y PCR.The plasmid isolated from these transformants were subjected to restriction digestion and sequencing.The cloned putative C. parapsilosis GBase (CparGBase) ORF (CPAR2_602270) was of 1104 bp (coding for a 367-residue polypeptide) with no introns predicted.

Heterologous expression of C. parapsilosis GBase (CpGBase)
The pET23a-CparGB plasmid bearing CparGBase ORF (CPAR2_602270 sequence) was separ atel y tr ansformed in E. coli BL21 (DE3) and E. coli Rosetta gamiB (DE3) pLysS.The growth and induction were carried out according to standard pr ocedur es (Sambrook and Russell 2001 ).Ureohydrolases, by and large, are metalloenzymes with a bimetallic center at their active site .T hey contain Mn[II], Co[II], Ni[II], or Zn[II] at the activ e site (Func k et al. 2022 ).The use of His-tag for the expression and purification of such enzymes would complicate the interpretation of activity data.Hence the use of His-tag was avoided.
Attempts to express CpGBase in S. cerevisiae , a yeast closely related to C. parapsilosis , were made .T he CpGBase ORF sequence was taken out from pET23a-CparGB (as Nde I-Xho I fr a gment) mov ed into a pBS vector (pBS-CparGB) and subsequently cloned as Eco RI-Xho I fr a gment in p426GPD (a plasmid used for constitutive protein expression in S. cerevisiae ; Figure S1, Supporting Information ).The resultant p426GPD-CparGB plasmid was transformed into S. cerevisiae 12T7cI strain ( car1::kanMX4, ura3 ); the p426GPD-GB (for expressing A. niger GBase) was also transformed in parallel, for comparison.The AnGBase cDN A w as mov ed as an Eco RI-Xho I fr a gment into p426GPD to obtain p426GPD-GB (Sar a gadam et al. 2019 ).Yeast transformed with empty p426GPD vector served as control.The transformants were selected based on uracil auxotrophy and were maintained on synthetic minimal media supplemented with 5 mM of AS or GB.
Gr owth pr ofiles of C. parapsilosis and S. cerevisiae transformants (Sc-Cp, expressing CpGBase or Sc-An, expressing AnGBase) on synthetic minimal medium a ppr opriatel y supplemented with differ ent nitr ogen sources (5 mM eac h of AS, AR, GB, GP, GABA, urea, or β-alanine) wer e compar ed both by spot assay on agar media as well as by growth curve in liquid broth.
The CpGBase ORF was cloned in a pCB-vector for its constitutiv e expr ession in A. niger .The Nde I-Not I fr a gment bearing this ORF was cloned in front of the constitutive A. niger citA promoter (in pCB XCGB; Kumar et al. 2015 ) to obtain pCB-PcitA Cpar.This plasmid was linearized with Sca I and used to transform A. niger protoplasts .T he transformants were single spored and characterized (by genomic PCR), for the integration of the plasmid.The transformants obtained along with the parent host strain ( A. niger NCIM 565) were spot inoculated (10 3 spores) on media containing differ ent nitr ogen sources (at 5 mM; ammonium nitrate, GB and GP) for growth comparison.

Assay of different ureohydrolases
T he arginase , GBase , and GPase activity in the cell-free extract w as assay ed using modified Archibald's method of urea detection as described earlier (Sar a gadam et al. 2019 ).One unit of arginase, GBase, or GPase activity is defined as the amount of enzyme required to produce 1 μmol of urea per minute under the standard assa y conditions .Specific activity is defined as units of enzymes per mg of protein.
The substr ate satur ations (with GB and GP) with enriched C. parapsilosis GBase (CpGBase) were performed using the 4-(dimeth ylamino)benzaldeh yde (DMAB) method optimized for GBase and GPase activity ( Supplementary Information S1 ; Figures S3 -S7, Supporting Information ).For the assay, the DMAB r ea gent was pr epar ed by dissolving DMAB (4%, w/v) in absolute ethanol and sulfuric acid (4%, v/v) and stored at 4 • C. T he assa y reaction includes phosphate buffer at 20 mM and GB (1-20 mM), and GP (1-40 mM).The reaction was initiated by adding the r espectiv e substrate and allowing the reaction to proceed for 20 min at 37 • C. The reaction was stopped by adding 750 μl of DMAB r ea gent (fr eshl y diluted with distilled water in a 1:2 ratio).These tubes were incubated at room temperature for 10 min, and absorbance at 420 nm w as recor ded.

Enrichment of CpGBase
To determine the kinetic parameters of CpGBase, it was enriched fr om the cell-fr ee extr act of Sc-Cp str ain ( S. cerevisiae expr essing CpGBase), using DEAE Sepharose and Superdex 200 columns sequentiall y.The DEAE Sephar ose column (manuall y pac ked, 20 ml bed v olume) w as pr e-equilibr ated with phosphate buffer.The desalted protein sample was loaded onto this and binding was done at a flow rate of about 0.3 ml/min.After washing with 6 column volumes of buffer (flow rate of 2 ml/min) the column was eluted at the same flow rate using a 0 to 1 M KCl linear gr adient.Fr actions (2 ml each) were collected and analyzed for CpGBase activity.The peak fractions from the DEAE step were chosen (based on activity and native PAGE analysis), pooled and concentrated by subjecting to 0%-80% AS satur ation.The pr otein pellet was dissolved in about 700 μl of buffer and proteins resolved on by gel filtration (Superdex 200, HiLoadTM 16/60 pr ep gr ade pr epac ked column, GE Healthcare).A 0.5-ml injection loop was used; the column was run at a flow rate of 1.0 ml/min at 25ºC.The fractions (1.0 ml, stored immediatel y at 4ºC) wer e assayed for CpGBase activity and analyzed on native PAGE.

Electr ophoretic pr ocedures
Pr otein electr ophor esis was performed in a Hoefer SE250 system according to the manufacturer's instructions.Both the na-tive PAGE (Davis 1964 ) and the SDS-PAGE (Laemmli 1970 ) were performed with minor modifications (Sar a gadam et al. 2019 ).
DNA electr ophor esis was performed in the horizontal gel electr ophor esis system (Bangalor e Genei, Bangalor e, India) following the manufacturer's guidelines .T he DN A w as separated in 1% a gar ose gel with 0.5 μg/ml ethidium bromide prepared in 1X TBE buffer.The gel picture was recorded under UV using the Geliance 1000 Imaging system (Perkin Elmer, Waltham, MA, USA).

Kinetic char acteriza tion of CpGBase
T he peak fractions , chosen based on the activity and gel profile were used as the enriched CpGBase for kinetic studies .T he enric hed fr actions wer e c hec ked for satur ation with GB (0.1-20 mM) and GP (2-40 mM) by DMAB assa y.T he data was plotted, and r espectiv e kinetic par ameters wer e determined.The enriched CpGBase from Sc-Cp strain (grown on synthetic minimal media supplemented with 5 mM GB) was used for inhibition studies .T he GBase and GPase activities were estimated by the modified Arc hibald's method (Sar a gadam et al. 2019 ).GABA and β-alanine were used at varying concentrations from 0 to 200 mM.

Protein estimation
Pr otein estimations wer e performed according to Bradford ( 1976 ), with bovine serum albumin as a r efer ence.

Homology modelling and bioinformatic analysis
The models to compare the ov er all structur es and the activ e sites of AnGBase (AHL44994) and CpGBase (OK067409) were generated using the Alpha-Fold.These models were compared for their loop regions at the active site with the Pseudomonas aeruginosa PAO1 GBase (3NIO) and GPase (3NIP) structures.

Identification of the yeast isolate that grows well on GP
The yeast isolate that efficiently catabolized both GB and GP was c har acterized in detail.It was identified as C. parapsilosis using internal transcribed spacer (ITS) sequencing.That sequence was deposited in NCBI GenBank under the accession number OK067409.The phylogenetic relationship of the isolated C. parapsilosis strain with related yeast strains is shown in Fig. 1 .The C. parapsilosis isolate, designated as the MEL404 strain, was deposited at the National Collection of Industrial Micr oor ganisms (NCIM), Pune, India, under the accession number NCIM 3689.

Profiling different ureohydrolase activities of C. parapsilosis NCIM 3689
T he yeast isolate , C. parapsilosis NCIM 3689, w as gro wn on AS, GB, GP, and AR and the r espectiv e cell-fr ee extr acts wer e assayed for arginase , GBase , and GPase activities .None of the three activities were detected when this yeast was grown on AS.Howe v er, all three activities were detected in GB and GP grown yeast cells.The GBase and GPase activities wer e strictl y induced in presence of GB or GP.Arginase activity was induced to some extent when GB or GP w as the sole nitrogen sour ce .T he specific activity ratios of GP ase:GBase, wer e similar in C. parapsilosis grown on GB and GP; suggesting that a single enzyme functions equally well, both as GBase and GPase in this yeast (Table 1 ).With the exception of P. aeruginosa PAO1 GPase, the GPase activity reported in all other cases is due to a br oadl y specific GBase (Yorifuji et al. 1982, Lee et al. 2011 ).Most broad specificity GBases display poor GPase activity   (Kumar et al. 2015 ) was used to search for putative GBase or GPase sequences in the CGD.The BLAST results sho w ed three putative ORFs (Table 2 ).CPAR2_602270 ORF (with a maximum identity of 50.4%) was chosen for PCR amplification.

Heterologous expression of C. parapsilosis GBase in E. coli
The pBLAST with AnGBase sequence as the query yielded three hits in the C. parapsilosis genome (from CGD) (Table 2 ).The putative C. parapsilosis GBase (CpGBase) ORF (CPAR2_602270) with the highest identity was 1104 bp long and was without introns.Based on this sequence, primers (CparGPF and CparGPR) were designed with Nde I and Xho I restriction sites for cloning this ORF.The putative CpGBase ORF was amplified using CparGPF and CparGPR primers and cloned in pET23aNat.Arg plasmid by replacing the arginase sequence between the Nde I and Xho I sites ( Figure S1, Supporting Information ).The resultant plasmid (pET23a-CparGB) was transformed in E. coli XL1 blue .T he ampicillin-r esistant tr ansformants wer e scr eened and c har acterized by colon y PCR and r estriction digestion.The positive transformants were further confirmed by sequencing the r ele v ant plasmids .T he pET23a-CparGB plasmid bearing CPAR2_602 270 sequence was transformed in E. coli BL21 (DE3).
The expression of a functional CpGBase in E. coli BL21 (DE3) could not be detected by the standard GBase assay under various IPTG induction and growth conditions .T he SDS-PAGE of the cell-fr ee extr acts also did not show the pr otein of expected size ( ∼40 kDa; a 367 aa polypeptide).Under similar conditions, howe v er, the AnGBase pr otein ( ∼50.4 kDa; a 422 aa polypeptide) was expr essed efficientl y (Fig. 2 ) and was also functional (Sar a gadam et al. 2019 ).Attempts were also made to express CpGBase in E. coli rossetta gamiB cells so as to rule out the possibility of the expr essed pr otein toxicity.Ho w e v er, no GBase activity or pr otein could be detected.

CpGBase expression in A. niger
The earlier growth showed that A. niger could efficiently utilize GB and β-alanine as the sole nitrogen sources, whereas GP was poorly utilized.Also, AnGBase sho w ed poor substrate specificity to w ar ds GP (Sar a gadam et al. 2019 ).Ho w e v er, the ov er expr ession of AnGBase in A. niger sho w ed impr ov ed gr owth on GP, suggesting that the growth on GP is limited by poor GPase activity of AnGBase.The heterologous expression of a functional CpGBase was anticipated to facilitate the growth of A. niger on GP .Accordingly , the CpGBase ORF was cloned in a pCB-vector under the constitutive PcitA to obtain pCB-PcitA Cpar ( Figure S2, Supporting Information ).This plasmid was linearized with Sca I and used to transform A. niger protoplasts (Kumar et al. 2015 ).After five passages on the selection medium (YDA supplemented with phosphinothricin) 18 stable transformants were obtained and four of them ((T3, T8, T13, and T14) were selected for further characterization by genomic PCR and by growth on different nitrogen sources.
The A. niger CpGBase transformants, XCGB13 str ain (ov er expressing AnGBase) and the parent A. niger NCIM 565 were spot inoculated on minimal media supplemented with different Nsources (5 mM of ammonium nitrate, GB or GP).All CpGBase transformants sho w ed compar able gr owth on ammonium nitr ate and GB, similar to that of XCGB13 strain and the parent strain (Fig. 3 ).While the XCGB13 str ain gr e w on GP plates (albeit slowly), none of the CpGBase transformants could do so.When the CpGBase tr ansformants gr own on minimal media (and their mycelial extr acts wer e anal yzed) none displa yed GBase activity.T he XCGB13 strain (as a positive control) did show GBase activity as expected (Kumar et al. 2015 ).

CpGBase expression in S. cerevisiae
The attempts to express putative CpGBase ORF in E. coli and A. niger were not fruitful.On the contrary, despite being a longer polypeptide (422 residues versus the 367 residues CpGBase), the AnGBase could be successfully expressed in both these organisms ( Kumar et al. 2015, Sar a gadam et al. 2019 ).This could be due to-(a) the codon bias between C. parapsilosis and these two expression hosts and/or (b) the instability of the expressed CpG-Base protein.The C. parapsilosis GBase sequence, although belonging to the CTG clade (P a pon et al. 2013 ), does not have a single CTG codon in the ORF.It is less likel y, ther efor e, that codon bias (residue changes to Ser in place of Leu) may be involv ed, while pr otein stability could be the cause for CpGBase nonexpression in both E. coli and A. niger hosts.Further, an attempt was made to express CpG-Base in S. cerevisiae (another yeast related to C. parapsilosis but that lacks endogenous GBase).The plasmid p426GPD, furnished with the constitutive gpd promoter, was used for this expression.The S. cerevisiae 12T7cI strain ( car1::kanMX4, ura3 ) is unable to utilize GB, GP and arginine and also provides for ura selection.The car1 bac kgr ound ensur ed that the major ur eohydr olase (i.e.ar ginase) is absent to facilitate-(a) clean screening of transformants and (b) monitoring the substrate specificity of expressed GBase through plate growth assa ys .Towards this , the plasmid p426GPD-CparGB was then transformed in S. cerevisiae 12T7cI strain; in parallel, the p426GPD-GB (for expressing A. niger GBase) was also transformed for comparison (see the section 'Methods').Yeast transformed with an empty p426GPD vector served as control.
The S. cerevisiae transformants bearing p426GPD-CparGB (abbr e viated as Sc-Cp str ain, expr essing CpGBase) sho w ed both GBase and GPase activity and also acquired the ability to utilize GB or GP, indicating that CpGBase ORF was successfully expressed in S. cerevisiae and that the expr essed pr otein was functional.Similarl y, Sc-An (expr essing AnGBase) and Sc-Ev (empty v ector contr ol) tr ansformants wer e also tested.The Sc-An str ain but not the Sc-Ev strain displayed GBase activity.The growth of the three S. cerevisiae transformants (Sc-Cp strain, Sc-An strain, and Sc-Ev strain) was compared by spot growth assays and through gr owth curv es. S. cerevisiae natur all y cannot utilize GB and GP as the sole nitrogen source.Inability of Sc-Ev strain to grow either on GB or GP plates is consistent with this observ ation.Yeast tr ansformants expressing AnGBase (Sc-An strain) or CpGBase (Sc-Cp str ain) wer e able to gr own on and utilize GB efficiently.Ho w ever, only the Sc-Cp strain could utilize GP as a nitrogen source, albeit poorl y (Fig. 4 ).A r elativ el y modest gr owth on GP could be due to poor transport/uptake of GP and the inability of S. cerevisiae (unlike C. parapsilosis ) to utilize β-alanine as a nitrogen source.It is possible that other putative enzymes in C. parapsilosis could impart better growth for this yeast on GP.It is known from literature that S. cerevisiae cannot utilize β-alanine (one product of GPase r eaction).Yet, S. cerevisiae gr owth on GP m ust exploit the other pr oduct ur ea as the av ailable nitr ogen source.Also, r elativ e r ates of urea utilization between the two yeasts could be different.Expressed CpGBase conferring growth advantage to S. cerevisiae on GP is ho w e v er clearl y established by this study.The growth profile of S. cerevisiae transformants (Sc-Ev, Sc-An, and Sc-Cp) in liquid culture is shown in Fig. 5 (A).All three strains utilized AS efficiently, and the growth reached the stationary phase within 48 h.The growth curve on AS for the isolated yeast ( C. parapsilosis NCIM 3689) was also comparable (Fig. 5 B).Ho w e v er, the gr owth of both the Sc-An and Sc-Cp strains was m uc h slo w er on GB than on AS, with the Sc-Cp str ain clearl y performing better.Both gr e w v ery poorl y on GP, while their r elativ e gro wth w as consistent with that observed on agar plates (Fig. 4 B).The growth difference between Sc-An and Sc-Cp, on GB as a sole source of nitrogen, is more prominent in submerged culture than that observed on agar plates.In contrast to the two S. cerevisiae F igure 4. Gro wth of Sc-An and Sc-Cp transformants on different nitrogen sources.(A) Saccharomyces cerevisiae transformants expressing AnGBase (Sc-An) and CpGBase (Sc-Cp) were spot inoculated (serial dilutions of 10 7 -10 3 cells/ml) on AS, GB, GP, AR, GABA, β-Ala, Urea, and YNB.All the nitrogen sour ces w er e pr esent at 5 mM and plates wer e incubated at 30 • C, and the ima ges wer e ca ptur ed e v ery 24 h.Yeast tr ansformed with an empty v ector (Sc-Ev strain) served as a control.(B) Growth data from days 1, 5, and 10 are highlighted, with growth on AS and Arg as controls.
F igure 5. Gro wth of C. parapsilosis and S. cerevisiae transformants in liquid culture.(A) The S. cerevisiae transformants Sc-Ev ( • • •), Sc-An (-), and Sc-Cp ( ) yeast cultures were grown (with 0.5 × 10 7 cells/ml as the inoculum) on different nitrogen sources (all at 5 mM), namely, AS( •), GB( ), GP( ), or AR( ) in liquid synthetic minimal medium.AR( ) is not plotted in panel (A) to avoid crowding, as there was no growth on this N source (the parent strain is car1 ).(B) Growth of C. parapsilosis NCIM 3689 (isolated strain) on different nitrogen sources in liquid culture (conditions and symbols are same as in A).Note: None of these organisms could grow on YNB alone.transformants, the isolated C. parapsilosis strain efficiently utilized both GB and GP and attained the stationary phase within 48 h.In fact, the growth of C. parapsilosis on all four nitrogen sources (including AS and AR) was compar able.Inter estingl y, the same CpGBase expressed in S. cerevisiae , while functional, is unable to pr ovide gr owth adv anta ge on GP to the host.This is not a r eflection of differences in enzyme expression level (significant levels of CpGBase, both as GBase and GPase activities, could be detected in the cell-free extracts of S. cerevisiae transformant).Hence, the observed poor growth could be due to poor transport/uptake of GP and the inability of the S. cerevisiae host strain (unlike the parent C. parapsilosis ) to utilize β-alanine as a nitrogen source.While the two organisms can effectively utilize GB, clearly, C. parapsilosis (NCIM 3689 isolate) is well endowed to assimilate GP also-a feature unlike A. niger (Saragadam et al. 2019 ).For these reasons, it w as w orth c har acterizing the pr operties of CpGBase in mor e detail.

Char acteriza tion of C. parapsilosis GBase
We noted earlier that the GPase activity of C. parapsilosis may be due to a single br oadl y specific GBase (Table 1 ).While C. parapsilosis could efficiently utilize GP through the activity of this enzyme, heter ologous expr ession of CpGBase (and not AnGBase!) did manifest some growth advantage to S. cerevisiae transformants.Only a handful of GBases have been studied so far and AnGBase is one of them (Sar a gadam et al. 2019 ).It was, ther efor e, of inter est to purify, kineticall y c har acterize and compar e the CpG-Base with AnGBase.Despite some efforts, a functional CpGBase enzyme could not be expressed in E. coli .It w as, ho w ever, possible to do so in the yeast bac kgr ound.Accordingl y, CpGBase was expressed and enriched 24-fold from cell-free extracts of Sc-Cp strain ( S. cerevisiae expressing CpGBase; see the section 'Methods'), using DEAE Sepharose and Superdex 200 columns and the peak fr actions wer e anal ysed on a nativ e-PAGE gel (Fig. 6 ).
The enriched fraction of CpGBase displayed both GBase and GPase activities .T he enzyme was used for GB (0.1-20 mM) and GP (2-40 mM) saturations and using a DMAB assay.Both GB and GP saturations sho w ed typical Mic haelis-Menten curv es (Fig. 7 ), and the V max /K M values (3.4 and 2.0, respectively) were similar.The kinetic features of CpGBase were compared with those of the betterc har acterized AnGBase, and these are listed in Table 3 .Clearly, AnGBase acts 30 times better on GB than GP, whereas CpGBase is just about twice as effective on GB than GP.Unlike AnGBase, CpG-Base is thus a broad specificity enzyme and acts equally well on both GB and GP.
Product inhibition analysis can throw further light on the specificity aspects of CpGBase .T her efor e, inhibition of the GBase and the GPase activities of CpGBase by their respective products (namel y, GABA fr om GB hydr ol ysis and β-alanine fr om GP hydr olysis) was studied.Similar product inhibition data for AnGBase was used for comparison.GABA inhibited both the GBase and the GPase activity of CpGBase equally w ell.Ho w ever, GAB A w as a strong inhibitor of the GPase activity of AnGBase than its GBase activity.Although β-alanine is a weak inhibitor when compared to GABA, it also showed a similar pattern of inhibitionbeing more effective on CpGBase (Fig. 8 ).Analysis of the inhibition data (Cheng and Prusoff 1973 ) was done to e v aluate the r espectiv e K I v alues for both GABA and β-alanine.A summary of inhibition results is given in Table 3 .The inhibition patterns again points to the broader substrate specificity of CpGBase than AnGBase.

Structural insights into C. parapsilosis GBase
Unlike AnGBase (a 422-r esidue pol ypeptide and highl y specific to w ar ds GB), the CpGBase is a smaller protein (367 residues)   and acts efficiently both on GB and GP.Identifying the residues critical for their substrate specificity is useful.It was, ther efor e, of interest to model and compare the two ureohydrolase structures .T he structural data on ureohydrolases is largely limited to arginases and currently only one GBase structure is available (Lee et al. 2011 ).This GBase is reported to be a 319-residue polypeptide that functions as a homotetramer.T herefore , the structural models of both AnGBase and CpGBase were built using the Robetta platform (an online tool that is unbiased to w ar ds an y single structur e while building the models).The two proteins were first analyzed for their amino acid sequence similarity ( Figure S8, Supporting Information ).The AnGBase model obtained was comparable with its overall structural fold ( α-β sandwich) and the active site geometry of the reported P. aeruginosa PAO1 GBase structure (Fig. 9 A).The AnGBase structure was then used to compare with that of modeled CpGBase structure.AnGBase sequence (with 422 residues) is longer than that of CpGBase (with 367 residues).Besides the sequences contributing to the common structural fold (the α-β sand wich), ad ditional sequence(s) in the AnGBase were found a wa y from the active site (Fig. 9 B).Like in the case of most known ur eohydr olase structur es, the active site in both AnGBase and CPBGase is largely built with flex-  ible loops (Fig. 10 ).The differences in the length and residues in the loop regions covering the active site account for the substrate entry and specificity.The corresponding loop regions of human arginase-I, P. aeruginosa PAO1 GBase, and P. aeruginosa PAO1 GPase wer e compar ed with those pr edicted in the AnGBase and CpGBase models .T he residues N130, M161, and Y157 in human arginase I, P. aeruginosa GBase , and GPase , r espectiv el y, wer e shown to be involved in substrate recognition (Alarcon et al 2006, Lee et al 2011 ).The corresponding loop residue was Y218 in AnGBase and S191 in CpGBase .T he r esidue with a r elativ el y small R gr oup (S191 in CpGBase) could possibly accommodate both GB and GP in the active site and may account for the broader substrate specificity of CpGBase (Fig. 10 ).These possibilities could be tested by suitable site-dir ected m uta genesis studies of the two enzymes.

Conclusions
The detailed kinetic c har acterization of CpGBase, sho w ed its br oader substr ate-specificity when compar ed to AnGBase.CpG-Base is a novel ureohydrolase , i.e .equally efficient with both GB and GP as substrates and is the first of its kind to be reported from fungi.A comparison of structural models derived from CpG-Base and AnGBase sequences points to their common structural folds with active sites defined largely by loop regions .Hence , predicting and/or defining substrate specificity of ureohydrolases thr ough doc king is a daunting task.Along with P. aeruginosa GBase (319 amino acids) and AnGBase (422 amino acids), the CpGBase (367 amino acids) now provides an excellent paradigm to explore the structures and active sites of ureohydrolases in general and GBases in particular.Ov er all, the pr esent work adds a ne w yeast enzyme to the list of ur eohydr olase superfamil y gr oup; unlike other enzymes of the family, this enzyme has wide substrate specificity and different catalytic properties.Finally, the broad substrate specificity of CpGBase, in combination with GP as its substrate, has the potential to serve as a novel nutritional selection marker for fungal transformations.

Figure 1 .
Figure 1.Phylogenetic analysis of the yeast isolate based on ITS1, 5.8S, 18S, and ITS4 rDNA sequences .T he arrow shows the position of this yeast isolate ( C. parapsilosis MEL404; deposited as NCIM 3689) in the phylogenetic tree .T he isolate is most closely related to C. parapsilosis strains .T he tree is rooted with S. pombe as an out-group and analysis details are given in the section 'Material and methods'.

Figure 6 .
Figure 6.Purification of CpGBase expressed in S. cerevisiae.Elution profiles of GBase activity ( •) and protein ( •) from the DEAE Sepharose column (A) and Superdex 200 gel filtration column (B) are shown.The native PAGE of four different CpGBase fractions (1, 2, 3, and 4 in the table) collected at differ ent sta ges of enric hment is also shown (C).The arr ow in (C) points to the potential CpGBase pr otein band based on its specific activity incr ease after each step of purification (activity-guided purification), as shown in the table.

Figure 7 .
Figure 7. Candida parapsilosis GBase substr ate satur ation kinetics .T he CpGBase enzyme assa ys for initial velocity analyses were performed in triplicate.The kinetic data is r epr esentativ e of three independent purifications.

Figure 8 .
Figure 8. Inhibition of AnGBase and CpGBase activities by the reaction products .T he GBase ( •) and GPase ( ) activity of AnGBase (-) and CpGBase ( ) was performed in the presence of varying concentrations of GABA (A) and β-alanine (B).The activity without an inhibitor was considered 100% and plotted by calculating the r espectiv e r esidual activity (%) with incr easing inhibitor concentr ation.Inhibition curv es ar e r epr esentativ e of three separate experiments.

Figure 9 .
Figure 9. Structural models and active site residues of AnGBase and CpGBase .T he P. aeruginosa PAO1 GBase (salmon) structure compared with AnGBase (c y an) model for the ov er all subunit structur e (A) and the activ e site with AnGBase r esidues labelled (B).The CpGBase (or ange) model compared with AnGBase subunit structure (C) and the corresponding active site (CpGbase residues labelled) (D).

Figur e 10 .
Figur e 10. T he active site loop regions of four different ureohydrolases.(A) The active site loop regions of CpGBase model (in green; top view) compared with those from human arginase-I (salmon), P. aeruginosa PAO1 GBase (y ello w), P. aeruginosa PAO1 GPase (magenta) and AnGBase (c y an).(B) The same active site viewed at 90 • angle with respective critical substrate recognition residues.

Table 2 .
Protein BLAST of C. parapsilosis genome queried with AnGBase sequence.

Table 3 .
A summary of kinetic parameters for AnGBase and CpGBase.