Structural analysis and functional evaluation of the disordered ß–hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis

Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1–110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region’s impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%–97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.

The 594-residue polypeptide that makes up the enzyme (BHT) has two distinct regions: the C-terminal domain, which is homologous to other glycosyl hydrolase family 1 (GH1) members, and the N-terminal section (residues 1-110), which is unique to BHT and has no homology to any known glycosyl transferases or β-glucosidases (Dagher et al., 2013;Dagher and Bruno-Bárcena, 2016).We previously showed the presence of an active 1-22 signal sequence with a membrane anchor signature inside the 110 N-terminal region using in silico analysis and subsequent functional studies using K. phaffii GS115 for secretion (Dagher and Bruno-Bárcena, 2016).In that study, the 1-22 signal sequence was replaced with MFα, which resulted in a 10-fold increase in the amount of secreted catalytically active rBHT in the culture broth compared to expression of full-length rBHT which remained membrane-bound.Surprisingly, the bulk of rBHT remained affixed to the K. phaffii GS115 membrane rather than being fully transferred to the medium (Dagher et al., 2013;Dagher and Bruno-Bárcena, 2016).
In silico analysis also revealed that the N-terminal region comprises regions of low complexity that have yet to be defined and characterized (Dagher et al., 2013;Dagher and Bruno-Bárcena, 2016).Furthermore, the crystal structures solved by Uehara et al., 2020 (HsBglA, PDB: 6M4E) (Uehara et al., 2020) and in this study (BHT, PDB: 7L74) showed a potential for an intrinsically disordered region (IDR) within the N-terminus.IDRs are flexible and extended protein segments known to lack organized secondary structure under physiological conditions.However, their biological function depends on this unstructured state (Uversky, 2019).Intrinsically disordered proteins (IDPs) exist in interchanging conformations rather than adapting well-defined structures as previously reviewed (Uversky, 2019).This is consistent with IDRs' functional advantages and ability to fold in response to partner contact or in a template-dependent manner (Darling and Uversky, 2018).
It has been demonstrated that proteins with large stretches of IDRs are essential elements for membrane interactions because these flexible areas allow for protein-protein or protein-lipid interactions, great selectivity and low affinities for key components of signal transduction cascades (Cornish et al., 2020).Additionally, membrane attachment constricts the protein's search space, consequently membrane localization can increase the effective concentration while simultaneously acting as a steric barrier to prevent interactions from occurring in solution (Cornish et al., 2020).
Proteome-wide investigations have shown connections between IDRs and several post-translational modifications (PTMs), including acetylation, methylation, and glycosylation (Gao and Xu, 2012).Previous studies by us and others in which E. coli was unable to express active rBHT suggested the critical importance of PTMs for appropriate folding and/or enzymatic activity (Ishikawa et al., 2005;Dagher and Bruno-Bárcena, 2016).One of the most important post-translational modifications of proteins is glycosylation, which primarily involves the attachment of glycans to the nitrogen atom of asparagine residues (N-linked) or to the hydroxyl oxygen of serine, threonine, or tyrosine residues (O-linked).Other important post-translational modifications of proteins include C-mannosylation, phospho-serine glycosylation, and glypiation (formation of GPI anchors) (Prabakaran et al., 2012;Darling and Uversky, 2018).In K. phaffii GS115, N-glycans form high-mannose-type heterogeneous oligosaccharides beginning with the addition of the core unit Glc 3 Man 9 GlcNAc 2 (Glc = glucose; GlcNAc = N-acetylglucosamine; Man = mannose) at asparagine in the recognition sequence Asn-X-Ser/Thr X≠P (Bretthauer and Castellino, 1999).N-glycosylation has been shown to influence enzymatic activity, stability, and cell surface expression as previously reviewed (Ge et al., 2018).
Further investigations into the intricacies of the structure of this enzyme are therefore needed to provide suggestions on how to enhance soluble secretion of rBHT.In this study we conducted a detailed kinetic analysis of rBHT variants lacking progressive portions of the IDR, in comparison to the full-length enzyme.To evaluate the impacts on protein secretion and enzyme activity, this study looked at modifications in the IDR length, N-glycosylation sites, and dimer stability.The results provide insight into the dynamics of the IDR related to enzyme secretion and localization of active rBHT generated by K. phaffii GS115.

Strains and media
The bacterial and K. phaffii GS115 strains used in this study are shown in Table 1.Bacteria were grown at 37 °C in Luria-Bertani (LB) medium with antibiotic ampicillin (100 μg/mL) (Thermo Fisher Scientific).Growth and maintenance of GS115 (Invitrogen Life Technologies, Thermo Fisher Scientific) was described previously (Dagher and Bruno-Bárcena, 2016).E. coli XL1-Blue was used as the cloning host (Agilent Technologies, Thermo Fisher Scientific).The plasmid pPIC9 (Invitrogen Life Technologies, Thermo Fisher Scientific) was used as cloning vector containing codon optimized Bht (rBht sequences) (GenBank accession number JF29828).

Plasmid constructions, expression, and purification of rBHT-truncated variants
All molecular biology protocols were carried out as previously described (Dagher and Bruno-Bárcena, 2016).Briefly, expression by K. phaffii GS115 was achieved by homologous integration of DNA fragments bearing rBht sequences, for example, coding for mutations and truncations.
As described above PCR amplicons were digested with XhoI-NotI and cloned into pPIC9 (Invitrogen Life Technologies, Thermo Fisher Scientific).DNA fragments from restriction enzyme digests were purified from agarose gels using QIAquick gel extraction kit (Qiagen, Hilden, Germany).All mutations were confirmed with restriction digests for detecting restriction sites in primers and by Sanger sequencing performed by the Azenta Life Sciences (USA) using primers JBB3, JBB4, 5′ AOX1, 3' AOX1 and α-factor (Table 2).

K. phaffii GS115 transformation and expression
K. phaffii GS115 was transformed with linearized plasmids as per the Invitrogen Pichia Expression Kit manual (Invitrogen, USA).Plasmid integration and Mut + phenotype in histidine positive colonies was confirmed by sequencing PCR products generated by primers 5′ AOX1 and 3' AOX1(Invitrogen Pichia expression kit).Single copy integration was confirmed as previously described (Dagher and Bruno-Bárcena, 2016).
Expression and purification have been described previously (Dagher and Bruno-Bárcena, 2016).Briefly, filtered culture media was purified using the ÄKTApurifier and HISTrap ™ HP Nickel column (GE Healthcare, Life sciences).The purified proteins were quantified by Bradford protein assay (Thermo Fisher Scientific) (Bradford, 1976).

SDS-PAGE and Western immunoblot analysis
Proteins were analyzed by SDS-PAGE using 10% resolving gels and visualized using Coomassie and silver stains (Bio-Rad, Hercules, CA).Immunoblots were probed with 1:10,000 dilution of anti-HIS antibody (GenScript, Piscataway, NJ) followed by 1:10,000 dilution of alkaline phosphatase conjugated goat anti-mouse antibody (GenScript, Piscataway, NJ).Detection was carried out with 1- Step ™ NBT/BCIP Substrate Solution according to manufacturer's instructions (Thermo Fisher Scientific).

Enzyme assays
Hydrolysis of o-nitrophenyl-β-D-glucopyranoside (ONP-Glc) was followed by measurement of absorbance at 405 nm for determination of β-glucosidase activity using the methods described previously (Dagher and Bruno-Bárcena, 2016).Briefly, cells were harvested by centrifugation (5,000 g at 4 °C), to separate soluble rBHT from membrane bound rBHT.The cells were then washed two times with 50 mM phosphate-citrate buffer (pH 5).Assays on soluble secreted and membrane bound rBHT were performed in a 50 mM phosphate-citrate buffer under optimal temperature of 42 °C and optimal pH 5 for 10 min.Reactions were stopped by the addition of an equal volume of 0.25 M sodium carbonate and the absorbance was measured at 405 nm.
Antibodies Antigen

Mouse anti-HIS 6XHIS GenScript
Substrates Abbreviation oNP-β-D-glucopyranoside ONP-Glc Sigma a Coding regions are capitalized, mutated nucleotides are bold and italicized, MFα, Saccharomyces cerevisiae alpha factor pre-pro secretion leader sequence.
The Michaelis-Menten constants (Km and Vmax) of 0.3 µg rBHT (at 42 °C) were determined by varying ONP-Glc from 0 to 10.4 mM in 50 mM phosphate-citrate buffer (pH 5) and measuring the initial reaction rate at 20 °C, 30 °C, 42 °C, and 55 °C.The kinetic constants at each temperature were determined with OriginPro 7.5 using nonlinear regression of the Hill equation with a Hill coefficient of 1.

N-glycosylation prediction
BHT N-and O-glycosylation site prediction was performed at the GlycoEP server (Chauhan et al., 2013).

Structural modeling programs
Structural figures and structural superimpositions were generated in PyMOL (http://www.schrodinger.com/pymol/)(Schrodinger, 2022).A homodimer is present in the crystal asymmetric unit; however, the monomer was considered for structural analysis.

Crystallization
rBHT (23-594) -HIS was further purified by gel filtration chromatography on a Sephacryl S-300 (GE Healthcare, Life Sciences) column equilibrated with 100 mM Tris pH 7.5, 200 mM sodium chloride, 1 mM dithiothreitol to reduce aggregates and concentrated to 6 mg/mL using Amicon ® Ultra 15, molecular weight cut-off 10,000 (Millipore Sigma) in 10 mM HEPES pH 7.5.Protein concentrations were determined by Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard.The crystals were grown by vapor diffusion using the sitting drop method.The crystals were grown using a crystallization solution made by mixing 1 µL (10 μg/μL) purified protein with 1 µL of precipitant solution (35% polyethylene glycol 4k, 0.1 M HEPES pH 7.5, 0.2 M calcium chloride) and equilibrating the drop against 0.5 mL of the precipitant at 22 °C-23 °C.Crystals usually appeared in less than a week.Prior to data collection, the crystals were soaked for 10 min in a cryoprotectant solution (35% polyethylene glycol 4k, 0.1 M HEPES pH 7.5, 0.2 M calcium chloride, 20% ethylene glycol) and then immediately flash-vitrified in liquid nitrogen.

Data collection, processing, and structure refinement
Single crystal diffraction data were collected at the Life Sciences Collaborative Access Team facility (Advanced Photon Source sector 21, Argonne National Laboratory (Lemont, IL, USA) on beamline 21G (Table 3).The data covered 360 °in 0.5-degree increments.The frames were integrated with XDS (Kabsch, 2010) and scaled with Aimless (Evans and Murshudov, 2013) in AutoProc (Vonrhein et al., 2011).The structure was solved by molecular replacement in PHENIX (Adams et al., 2010) using a homology model generated by RaptorX (Källberg et al., 2012).The structure was rebuilt and refined with PHENIX and then optimized with PDB-REDO (Joosten et al., 2014).Coot (Emsley et al., 2010) was used to add and to optimize individual residues, posttranslational modifications and ligands.

Results
Crystal structure of rBHT shows hallmarks of intrinsic disorder in the N-terminal domain When expressed by K. phaffii GS115, the rBHT variant (rBHT (23-594) -6XHIS) is functionally independent of its location either associated with the membrane or soluble (Dagher et al., 2013;Dagher and Bruno-Bárcena, 2016).To gain insights into the structure-function characteristics, we used soluble rBHT (23-594) -6XHIS to solve the crystal structure.Data processing and refinement statistics are presented in Table 3 and the final model was deposited in the Protein Data Bank (PDB: 7L74).The structure was solved by molecular replacement at a resolution of 2.25 Å and the asymmetric unit contains two molecules of rBHT (23-594) -6XHIS and the value V m was estimated to be 2.46 Å 3 .Da -1 .
rBHT folds into two domains, the N-domain, and the C-domain.The structure for enzymatic activity in the C-terminal region is composed of a sugar-binding catalytic domain organized in a (α/β) 8 TIM barrel that stretches from residue 116 to residue 547 (BHT, PDB: 7L74).Eight parallel β-strands comprise the core BHT (α/β) 8 barrel, which is coupled to eight external α-helices that is common to Glycoside Hydrolase Family I (GH1) members (Henrissat et al., 1995;Glycoside Hydrolase Family, 2012).
The initial portion of the N-domain (residues 23-53) upstream of the carboxy GH1 domain, lacked electron density and could not be modelled indicating the presence of a potential N-terminal intrinsically disordered region (IDR).Additional support for the IDR within the N-terminal domain comes from a previously solved crystal structure (HsBglA, PDB: 6M4E) (Uehara et al., 2020).HsBglA and rBHT crystal structures show minor structural differences with an RMSD across all Cα pairs of 0.22 Å.

BHT N-terminal IDR composition
The relevance of the disordered regions in membrane lipid association and interactions of membrane associated proteins can only be understood by examining the properties of the interacting environment (Mohammad et al., 2019;Cornish et al., 2020;Csizmadia et al., 2021).This can be complicated by the multiple interactions and functions exhibited by disordered regions (Theillet et al., 2013;Uversky, 2019) and their ability to fold upon contact in a template-dependent manner or with specific ligand partners (Bürgi et al., 2016).
To make the IDR structure accessible for systematic analysis, IDR boundaries and PTM predictions needed to be made to reveal incomplete regions, which is particularly important for IDR analysis as described and shown below (Figure 1).This compelled us to perform a series of in silico structural predictions by using five available prediction tools over the full length of BHT (Figure 1).By combining different disorder predictors we expect to reinforce the reliability of the predicted regions since they use different definitions of disorder (Lieutaud et al., 2016).For example, PSIPRED and Globplot methods were employed to strengthen the lack of secondary structure and globular domains in the IDR region.Upon comparison, the disorder datasets derived from Phyre2, IUPred2A, DISOPRED3, Globplot Disorder and PONDR indicate probable disorder boundaries throughout the unique 1-110 Nterminal region and a common overlapping boundary at residues 52-53 (Figure 1), in agreement with PDB: 7L74 N-terminal boundary lacking electron density.
IDRs often contain a substantial degree of post-translational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acylation, and others that mediate potential interactions with high specificity (van der Lee et al., 2014;Cornish et al., 2020).For instance, phosphorylation can stabilize the tertiary structural organization of the IDR while enhancing and stabilizing its binding to the protein's ligand (Gsponer et al., 2008;Nishi et al., 2011).Bioinformatic analysis has suggested that this function is tunable by PTMs and correlated with a high content of serine, threonine, glutamine and asparagine (Chuang et al., 2020).

Expression and secretion of truncated N-terminal rBHT variants by K. phaffii GS115
Biologically important disordered regions have also been known as N-terminal fusion carriers to promote protein folding, act in folding quality control and thus enhance protein solubility.Our approach was to utilize the in silico analysis (Figure 1B) to perform progressive and selective deletions of the predicted IDR and to determine their impact on soluble secretion of catalytically active rBHT.Predictions were also made by the algorithm DisPhos1.3(DEPP) that uses disorder information to help improve and discriminate between phosphorylation and non-phosphorylation sites (Materials and Methods).Furthermore, the accuracy of DEPP reaches 76.0±0.3%, 81.3±0.3% and 83.3±0.3% for serine, threonine, and tyrosine respectively (Iakoucheva et al., 2004;Ingrell et al., 2007).
Methanol induced protein expression of each variant by K. phaffii GS115, for both membrane associated and soluble enzymes were evaluated as previously described (Dagher and Bruno-Bárcena, 2016).
Under our experimental conditions, the results showed undetectable amounts of soluble, or membrane associated active protein when residues downstream of the IDR were removed (Table 4).To further evaluate whether bioactive rBHT (82-594) -HIS, rBHT (95-594) -HIS and rBHT (103-594) -HIS variants, were produced and secreted in low amounts, inductions of the corresponding cell lines were performed, and culture broth was concentrated 100-fold.However, neither soluble nor cell-associated rBHT from those variants had any enzymatic activity.This may be caused by ineffective secretion or destruction of the protein molecules that were not secreted.
The combined results strengthen a new finding that the BHT IDR by itself is not directly responsible for enzymatic activity or membrane interactions (Table 4).

Kinetic parameters of secreted rBHT variants
Since the initial analysis of truncated variants revealed greater rBHT (57-594) -HIS titers (Table 4), we investigated if the structural organization exhibits kinetic biases or, more specifically, whether the IDR more broadly affects protein kinetics.
Active soluble secreted rBHT variants were functionally assessed by conventional kinetic measurements after being purified to homogeneity utilizing a C-terminal 6xHistidine epitope and Nickel affinity chromatography.Examination of the isolated rBHT variants by SDS-PAGE separation under reducing conditions showed the proteins were essentially homogenous (data not shown).
The kinetic parameters of each active secreted soluble variant, rBHT (23-594) -HIS, rBHT (32-594) -HIS, rBHT (54-594) -HIS, and rBHT (57-594) -HIS were investigated.To obtain a full kinetic picture, an important parameter evaluated was the impact of temperature on enzymatic activity.Therefore, assays were performed at the optimum temperature for rBHT (23-594) -HIS of 42 °C (Dagher and Bruno-Bárcena, 2016), below 42 °C (20 °C and 30 °C) and above 42 °C (55 °C) (Figure 4).The results of the respective kcat/km for all four truncated enzyme variants indicate a temperature optimum of 42 °C (Figure 4).At each temperature tested all enzyme-truncated variants maintain a similar affinity for the substrate ONP-Glc (Km) and turnover activity (kcat) therefore confirming that truncations within the IDR do not play a role in the catalytic integrity of rBHT.

Are N -glycans essential for secretion?
Proteome-wide investigations have revealed relationships between IDRs and several PTMs, including acetylation,  methylation, and glycosylation (Dunker et al., 2013).Extensive in silico analysis revealed there are only 4 predicted N-linked glycosylation sites out of a possible 19, and none were discovered in the 110-residue N-terminus that contains the IDR (Figure 1).Investigations have indicated that glycosites are found mostly in structured sections, some distance from the disordered stretches, which is consistent with our findings (Singh et al., 2018;Goutham et al., 2020).All four N-glycosylation sites are in highly conserved glycosylation consensus sites (Asn-X-Ser/Thr X≠Pro) and nearby residues in the crystal structures of rBHT (23-594) -HIS (BHT, PDB: 7L74 and HsBglA, PMB: 6M4E), indicating a high likelihood of functionally relevant glycosylation at those positions N289LTY, N297STS, N431QSD, and N569QSD.
We purified each N-glycosylation mutant by Nichromatography and confirmed that the variants had similar specific activities as the non-mutated forms (Table 4).Therefore, altering the N-glycosylation site interfered with secretion but did not alter the activity of the variants.The value of cell density (OD 600nm ) reached by the recombinant strains (Enzyme Source) after methanol induction was used to normalize the secreted soluble and membrane-bound activities.
The maximum cell densities obtained were between 60 and 75 OD 600nm .The results are mean values for three measurements of enzyme activity and standard deviation (SD)."ND" indicates enzyme activity was not detected.

Interface analysis
rBHT was crystallized in the C2 space group with two molecules per asymmetric unit, suggesting a possible dimer (Table 3).The same dimer is found in 6M4E, though in 6M4E a crystallographic axis of 2-fold symmetry runs through the dimer leading to only one molecule per asymmetric unit (Uehara et al., 2020).To distinguish between significant crystal interface interactions and artifacts of crystal packing (Krissinel, 2010) we used the program PISA (Protein Interfaces, Surfaces, and Assemblies) which calculates interface stability and entropy of dissociation to identify stable chemical contacts (Krissinel and Henrick, 2007).Analysis of PDB: 7L74 revealed a buried surface area of 7,803.5Å 2 between molecules A and B. The interface between molecules A and B has the largest negative Δ i G (−21.3 kcal/mol) so it is predicted to be the strongest and with dissociation energy ΔG diss (23.8 kcal/mol) and corresponded with the experimental results shown below.Residues in Loops C (yellow) and D (orange) contribute to most of the protein-protein interaction formed between the monomers (Figure 5).The residues involved in this interaction include hydrogen bond formation and salt bridges as shown in Figure 5B and depicted as yellow, orange, and gray sticks (Figure 5A).

Influence of N-terminal deletions on rBHT dimerization
To gain further insights into the oligomerization properties of BHT, we performed small X-ray scattering (SAXS) analysis (Figure 6A).Guinier and P(r) analysis was performed using PRIMUS and GNOM, respectively (Svergun, Konarev et al., 2003).D max values were manually chosen in GNOM to optimize the P(r) calculation (Figure 6B).These D max values are approximate to ~±2-3 Å.Molecular mass were calculated using the method described by Rambo and Tainer (Rambo and Tainer, 2013).The data are presented in Figure 6C.The molecular mass determined from SAXS (~169 KDa) confirmed that rBHT forms a dimer in solution (Figure 6C).The R g and D max of the dimer in solution are 39 Å and 124 Å, respectively.As stated above, the X-ray crystallographic structures (PDB: 7L74, 6M4E, 6M4F and 6M55) also suggest that rBHT forms a dimer.The R g and D max of the PDB: 7L74 crystallographic dimer (molecule A and molecule B) calculated using Crysol are 34 Å and 110 Å, respectively (Svergun et al., 1995).These values are in agreement with the experimental SAXS data.The disordered N-terminus led to a more expanded dimer in solution, and we conclude that rBHT (23-594) -HIS likely functions as a dimer.The SAXS experimental data have been deposited in the Small-Angle Scattering Biological Data Bank (SASBDB) (https://www.sasbdb.org/)under accession codes SASDN57 for rBHT (23-594) -HIS, 1 mg/mL and SASDN67 for rBHT (23-594) -HIS, 4 mg/mL.

Discussion
Here, we investigated whether the presence of the distinctive N-terminal intrinsically disordered region (IDR) and/or putative posttranslational modifications in the GH1 C-terminal domain affect the amount of secreted active BHT.These results confirm that the rBHT IDR is not essential for activity or drive proteinmembrane interactions.
Because the crystal structure did not provide any observable electron density at the N-terminal residues 23 to 53, IUPRED2A was used to predict the C-terminal border of the disordered region at amino acid 56.Deletion variants were generated based on the expected disordered portions until all 56 N-terminal residues were removed.Although native BHT is a membraneassociated protein, all rBHT variations partitioned between soluble secreted and cell membrane associated forms.Furthermore, soluble secreted enzyme variants that were shorted up to residue 56 displayed comparable catalytic properties.Although additional N-terminal deletions variants were not detected, it is possible that their removal affected rBHT's stability or secretory pathway.Disordered regions can be discriminated from ordered ones based on the amino acid sequence (Garner et al., 1998;Darling and Uversky, 2018) and in most cases, disordered proteins are less evolutionarily conserved but rather their disordered structure has been maintained (Brown et al., 2011).Previously reviewed data indicated that low sequence complexity, high net charge, and low concentration of hydrophobic residues are a hallmark of disordered protein regions employed for interactions with lipid bilayers (Theillet et al., 2013;Cornish et al., 2020).However, a significant elemental preference for disorder-promoting residues reported in classical IDRs is called into doubt by the high proportion of hydrophobic residues in the BHT IDR (35.6%), placing it within the category of molecular recognition features (MoRFs) (Theillet et al., 2013;Yan et al., 2015).
Beyond the secretion signal sequences chosen, several other factors also govern protein secretion.For instance, the release of heterologous proteins depends on N-glycosylation, a posttranslational modification involved in protein folding in the ER (Skropeta, 2009).It was therefore crucial to conduct additional research on the relationship between rBHT N-glycosylation and enzymatic properties to assess the stability, activity, and even secretion of the enzyme.However, not all polypeptides with predicted N-glycosylated sequons are glycosylated in vivo.Finding the locations of the N-linked glycosylation sites in the C-terminal GH1 domain was made easier by solving the crystal structure of rBHT (23-594) -HIS.No N-glycosylation sites were predicted in the IDR region, even though algorithms were useful at predicting O-glycosylation sites within the IDR (Figure 1).In this study, in vivo analyses were primarily used to evaluate the impact of eliminating a putative glycosylation site on expression, secretion, and activity.Although it appears that glycosylation is not necessary for enzymatic activity, the significant decrease in overall protein secretion observed for each of the four variants suggested that glycosylation may provide protection by increasing protein stability, shielding exposed hydrophobic surfaces, reducing proteolysis, and even increasing solubility.When associated to the membrane, BHT must be conformationally flexible, whereas when unconnected to the membrane, it must be stable.Given that rBHT homodimer activity and stability when expressed by K. phaffii GS115 are independent of the N-terminal 56 amino acids, it is possible that elements (PTMs) in addition to unique amino acids within the catalytic domain may serve as a handle for specific catalytic advantages in preserving the active enzyme.The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This work was supported by the Department of Plant and Microbial Biology, the Office of Research Commercialization, and the Chancellor's Innovation Fund (1,108)

FIGURE 5
FIGURE 5 Structural organization of rBht (23-594) -HIS dimer interface.(A) A ribbon representation of rBht (23-594) -HIS dimer highlighting loops (A; blue, B; green, C; yellow, D; orange) surrounding the active site is shown on the left.The inset on the right shows the dimer interface in greater detail with predicted salt bridges and H-bonds as black dashed lines.(B) Types of interface bonds and distances.The TRIS molecules occupying the −1 subsite is represented as a magenta stick.NAG are shown as green sticks.Ca 2+ ion is represented as a black sphere.The figure was produced using PyMOL (https://pymol.org/2/)(Schrodinger, 2022).PDBePISA (https://www.ebi.ac.uk/pdbe/pisa/ picite.html)was used to predict interface bonds and distances(Krissinel and Henrick, 2007).

FIGURE 6
FIGURE 6 SAXS data for rBHT (23-594) -HIS at 1 mg/mL and 4 mg/mL.(A) SAXS data are shown on a log-log plot (left).I(Q) is in arbitrary units.(B) P(r) curve calculated from the SAXS data are normalized to a maximum height of 1.0.(C) Solution scattering parameters zero-angle intensity I 0 , radius of gyration R g , and maximum dimension D max and SAXS-calculated molecular weight for rBHT (23-594) -HIS at 1 mg/mL and 4 mg/mL.
(2018-2092 to JB-B) at North Carolina State University; Protein crystallization and data generation used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357; Use of the Life Sciences Collaborative Access Team (LS-CAT) Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (085P1000817 to BE).

TABLE 1 (
Continued) Strains and plasmids used in this study.

TABLE 2
Primers, antibodies, and substrates used in this study.

TABLE 3
Data collection and refinement statistics of the structure of rBHT (23-594) -HIS at pH 7.5.
a R merge = hkl I | I i (hkl) −<I(hkl)> | hkl i I i (hkl), where I i (hkl) is the intensity of the ith measurement of reflection hkl, including symmetry-related reflections, and <I(hkl)> is their average.bRwork = h i ||Fo| − |F c ||/ |F o |. cR free is Rwork for ~5% of the reflections that were excluded from the refinement.

TABLE 4
Normalized enzyme activity comparison of (A) soluble versus (B) membrane bound secreted protein variants.