Relief of Xylose Binding to Cellobiose Phosphorylase by a Single

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Relief of xylose binding to cellobiose phosphorylase by a single distal mutation Kulika Chomvong, Eric Lin, Michael Blaisse, Abigail E. Gillespie, and Jamie H.D. Cate ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00211 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

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ACS Synthetic Biology

Conformational Change

ACS Paragon Plus Environment

Distal Mutation


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Chomvong et al. 1

Relief of xylose binding to cellobiose phosphorylase by a

2

single distal mutation

3 4

Kulika Chomvong1, Eric Lin2, Michael Blaisse3, Abigail E. Gillespie4 and Jamie H.D.

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Cate3,4,5,*

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1

8

94720;

9

2

Department of Plant and Microbial Biology, University of California, Berkeley, CA

Department of Chemical and Biomolecular Engineering, University of California,

10

Berkeley, CA 94720;

11

3

12

4

13

94720;

14

5

15

94720

Department of Chemistry, University of California, Berkeley, CA 94720; Department of Molecular and Cell Biology, University of California, Berkeley, CA

Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA

16 17

*

Corresponding author (email: [email protected])

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1


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Chomvong et al. 23 24

Abstract Cellobiose phosphorylase (CBP) cleaves cellobiose–abundant in plant biomass–

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to glucose and glucose 1-phosphate. However, the pentose sugar xylose, also

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abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse

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reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a

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single hydroxymethyl group relative to glucose, from binding to the CBP active site

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poses a spatial challenge for protein engineering, since simple steric occlusion cannot

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be used to block xylose binding without also preventing glucose binding. Using

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CRISPR-based chromosomal library selection, we identified a distal mutation in CBP,

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Y47H, responsible for improved cellobiose consumption in the presence of xylose. In

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silico analysis suggests this mutation may alter the conformation of the cellobiose

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phosphorylase dimer complex to reduce xylose binding to the active site. These results

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may aid in engineering carbohydrate phosphorylases for improved specificity in biofuel

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production, and also in the production of industrially important oligosaccharides.

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Keywords: cellobiose, phosphorylase, xylose, inhibitor, protein engineering

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Chomvong et al. 40

Enzymes are the crucial workhorses for most chemical conversions in biology.

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Their precise activity and specificity are fine-tuned through evolution of the polypeptide

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chain as part of a complex regulatory network in living organisms, ensuring survival in

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challenging environments. Given their efficiency, many enzymes produced biologically

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have been used in industrial settings to catalyze chemical reactions on a scale much

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larger than their native environment. However, the conditions in which they operate

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industrially likely do not match those of the endogenous cells in which they evolved, for

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example involving different temperatures, pH values, and other chemicals. Exposure to

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novel conditions may directly affect the enzyme efficiency and even render them

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inactive.

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Cellobiose phosphorylase (CBP) is used by many organisms as a means to

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consume cellobiose released from plant cell walls as a carbon source, often in

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anaerobic conditions. CBP is an energy efficient enzyme capable of cleaving cellobiose

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to glucose and glucose 1-phosphate (G1P) in the presence of inorganic phosphate 1,2.

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Unfortunately, its activity is greatly compromised in the presence of xylose. Xylose is a

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known mixed-inhibitor of the cellobiose phosphorylase (CBP) enzyme, reducing CBP’s

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apparent affinity for cellobiose and reducing its apparent maximal rate 3. In addition,

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xylose and G1P can be used as substrates by the reverse CBP reaction, producing 4-

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O-ß-D-glucopyranosyl-D-xylose (GX), which is observed in vivo when xylose is present

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during cellobiose consumption 3,4 (Figure S1). This is problematic because cellobiose

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and xylose generally co-exist as components of plant biomass during its deconstruction.

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Thus, improving the use of CBP to be tolerant to the presence of xylose is critical for its

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industrial use.

3


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Chomvong et al. 63

Although pathway engineering could partially alleviate the negative impact of

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xylose on cellobiose conversion, it would be preferable to improve the enzymatic

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properties of CBP directly. However, this particular protein-engineering problem is quite

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challenging. The fact that CBP produces GX from glucose 1-phosphate and xylose by

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the reverse reaction 3 directly implies that xylose is capable of binding to the CBP active

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site. To react, it must bind to the same pocket as the glucose product from the forward

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CBP reaction 5. To relieve xylose inhibition, the enzyme would need to be modified so

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that xylose can no longer bind to the active site, while still allowing glucose to be

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released from the enzyme complex. Since glucose is released before glucose 1-

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phosphate 6, it is crucial for xylose not to bind to the glucose pocket before glucose 1-

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phosphate is released, as the reverse reaction of GX formation has a favorable Gibbs

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free energy 2,7. Considering how xylose is identical to glucose apart from the removal of

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a single hydroxymethyl group, using steric constraints to prevent xylose binding would

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probably prevent glucose binding, as well.

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To identify CBP variants that are less affected by xylose, a chromosomal CBP

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library was created using error-prone PCR and the Multiplex CRISPR (CRISPRm)

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system 8. This approach is beneficial because it circumvents variable copy number

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effects observed in a plasmid-based expression system. Mutated Saccharophagus

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degradans cbp was assembled at the URA3 locus (ura3::PPGK1-cbp-TADH1) into a S288C

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diploid strain expressing an improved cellodextrin transporter (lyp1::PTDH3-cdt1 F213L-

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TCYC1) 2. The library was pooled and subjected to selection in liquid media containing

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1% cellobiose and with 2% xylose as a competitor. High concentrations of xylose in the

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cellobiose media would be expected to act as a selection pressure, favoring cbp

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Chomvong et al. 86

mutants capable of overcoming mixed inhibition by xylose and/or reducing GX synthesis

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by CBP.

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Of 10 large colonies picked from the selection, strain CX9 showed improved

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aerobic growth in 1% cellobiose plus 2% xylose (Figure S2). The cbp allele in the CX9

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strain (cbpCX9) had 7 mutations—Y47H, K256M, E270D, I384L, N488D, N578S and

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Y631F—located throughout the protein structure (Figure 1A). To validate that the

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improved phenotype was not a consequence of background mutations, cbpCX9 was

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reintroduced into the initial strain. In aerobic conditions, the strain expressing cbpCX9

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showed improved growth and cellobiose consumption in comparison to the strain

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expressing wild-type CBP (Figure 1B).

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Of the 7 mutations present in cbpCX9, Y47H was necessary and sufficient to

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replicate nearly all the improved consumption phenotype of cbpCX9 (Figure 2A, Figure

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S3). While the cellobiose consumption profile of cbp Y47H was similar to that of cbpCX9,

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its extracellular xylose and GX concentrations were intermediate between those of

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strains expressing wild-type CBP or cbpCX9 (Figure 2). The cbp mutants had higher

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extracellular xylose concentrations consistent with the lower secreted GX

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concentrations at all time points. These results suggest that xylose did not interact with

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cbp mutants as well, decreasing GX formation, thereby improving overall cellobiose

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consumption in the presence of xylose. However, the difference between the two cbp

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mutants, evident through the xylose and GX concentrations, suggests that the other

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mutations in addition to Y47H in cbpCX9 also contribute to the improvement of the cbpCX9

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strain.

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Chomvong et al. 108

Next, we studied the in vitro effects of cbp mutants. Xylose competition and GX

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synthesis assays were performed using purified cbp proteins (Figure 3A, Figure S4).

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Xylose acts as a mixed inhibitor of the cellobiose phosphorolytic forward reaction 3,

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resulting in a decrease in Vmax,app and an increase in Km,app. For the cbp mutants, the

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decrease in Vmax,app in the presence of xylose was attenuated by 60-70%. However, a

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substantial rescue of the increase in Km,app in the presence of xylose was only observed

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in cbpCX9 (40%). The increase in Km,app of cbp Y47H remained elevated, comparable to

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that of wild-type CBP. These results suggest that, unlike cbpCX9, Y47H only relieved

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xylose inhibition at the level of Vmax,app but not Km,app. The difference in their response to

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xylose in terms of Km,app might explain the intermediate phenotype of strains expressing

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cbp Y47H in the presence of xylose.

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To test the effect of cbp mutations on the reverse reaction with xylose, end-point

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GX synthesis assays were performed (Figure 3B, Figure S4). The amount of GX

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synthesized was normalized by the amount of cellobiose synthesized by the same

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enzyme variant, with either xylose or glucose provided as one of the substrates,

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respectively. Wild-type CBP synthesized 5 times more GX than the cbp mutants,

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suggesting that the mutations decrease the ability of CBP to carry out the reverse

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reaction with xylose as a substrate, consistent with the lower GX concentrations

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observed in the media in growth assays.

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To test the generality of the Y47H mutation in improving cellobiose conversion in

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the presence of xylose, the highly conserved Y47 (Figure 4A) was mutated to histidine

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in a Saccharophagus degradans CBP (SdCBP) homolog from Cellulomonas gilvus

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(CgCBP, 63% identity). Notably, similar in vivo and in vitro effects were observed with

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Chomvong et al. 131

CgCBP when compared to SdCBP. In the presence of xylose, cellobiose consumption

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of yeast expressing Cgcbp Y47H improved in comparison to the strain expressing wild-

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type Cgcbp (Figure 4B). Extracellular xylose in cellobiose consumption experiments

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with the strain expressing Cgcbp Y47H was also higher than that of the wild-type Cgcbp

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strain, corresponding to the lower amount of GX secreted (Figure S5). In the xylose

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competition assay, xylose had no effect on Vmax,app and Km,app of CgCBP Y47H, while

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wild-type CgCBP exhibited the expected decrease in Vmax,app and increase in Km,app, as

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observed for SdCBP (Figure 4C, Figure S6). Although the difference of the Y47H

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mutation on Km,app of SdCBP and CgCBP remains to be investigated, this difference

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does not seem to affect cellobiose consumption in vivo. In the end-point GX synthesis

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assay, wild-type CgCBP synthesized 2.6 times more GX relative to cellobiose than the

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CgCBP Y47H mutant (Figure 4D, Figure S6). Together, these results suggest that the

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improved phenotypes associated with the Y47H mutation might be conserved generally

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among CBP homologues.

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To understand the effect of the Y47H mutation on the structure of CBP, the

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crystal structure of Cellulomonas uda CBP (CuCBP) in complex with cellobiose

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(PDB:3S4A) 9 was used for modeling the Y47H mutation. Although the mutation is distal

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to the CuCBP active site (Figure 5A, Figure S7A), studies have revealed that distal

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mutations can greatly contribute to enzyme kinetics as they may change protein

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conformations and that they are part of the protein functional residue network 10,11. In

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terms of the amino acid replacement at Y47 position, one of the possible rotamers of

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histidine at position 47 shows minimal van der Waals overlap with surrounding protein

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side chains (Figure 5B). A surface view of the structure reveals cellobiose to be buried

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Chomvong et al. 154

inside CBP, with the Y47H mutation situated on the surface of the structure (Figure 5C,

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Figure S7B). Electrostatic calculations reveal that the Y47H position resides at the edge

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of a positively-charged elongated pocket, surrounded by negatively-charged residues,

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formed between two CBP units, suggesting that the dimer interface contributes to the

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enzyme catalytic mechanism. A similar electrostatic distribution exists when using the

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crystal structure of CgCBP in complex with glucose and SO4 (PDB:2CQS) 12 for the

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analysis.

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Mutation from a moderately polar tyrosine to a positively-charged histidine on the

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complex surface likely enhances salt bridge formation with nearby negatively-charged

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residues, possibly modulating the conformational dynamics of the CBP dimer, and

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thereby improving the apparent kinetics and reduced GX formation. Improved cellobiose

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production by CBP Y47H in the reverse reaction, in the presence of xylose, supports

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this hypothesis. We speculate that the Y47H mutation could modulate conformational

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dynamics between the subunits required for activity that partially blocks xylose entrance

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to the active site. It could also induce conformational changes that reduce xylose

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allosteric inhibition of CBP. For example, the Y47H mutation may decrease xylose

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binding to one active site that inhibits the catalytic activity of the other active site in the

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dimer. Structural studies of the CBP Y47H compared to wild-type CBP by X-ray

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crystallography or nuclear magnetic resonance spectroscopy may help identify the

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conformational changes caused by the Y47H mutation.

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In this study, we identified a single mutation in CBP, which helps alleviate

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inhibition by xylose. This mutation, located distal from the enzyme active site, provides a

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rare opportunity to determine how protein conformations can prevent small molecule

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Chomvong et al. 177

binding yet allow a larger molecule to occupy the same pocket within the enzyme. It

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may also shed light on how specificity is achieved more broadly in the carbohydrate

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phosphorylase family of enzymes now being explored for industrial applications,

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including artificial oligosaccharide production 13.

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Materials and Methods

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Yeast strains, selection and anaerobic fermentation

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All strains used in this study are listed in Table S1. S. cerevisiae S288C diploid

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strain was used for chromosomal library selection using the CRISPRm system 8. First,

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cellodextrin transporter CDT-1 with a F213L mutation 2 was introduced at the lyp1 locus

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(lyp1::PTDH3-cdt1 F213L-TCYC1). Then, codon optimized cellobiose phosphorylase from

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Saccharophagus degradans was subjected to mutation using the error-prone PCR

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GeneMorph II Random Mutagenesis Kit (Agilent Technologies) and introduced at the

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URA3 locus (ura3::PPGK1-cbp-TADH1). For the PCR reaction, a medium mutation rate

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program, using 250 ng of the initial target for the mutation frequency of 4.5 – 9

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mutations/kb, was performed with the manufacturer’s suggested PCR program (95 °C

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for 2 minutes – 95 °C for 30 seconds, 50 °C for 30 seconds, 72 °C for 4 minutes for 30

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cycles — finish at 72 °C for 10 minutes). The library was pooled and selected in liquid

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oMM media 14 containing 2% xylose (Fisher Scientific) and 1% cellobiose (Sigma-

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Aldrich). After 3 days, the selection culture was plated on minimal media plates

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containing 2% xylose and 1% cellobiose to screen for larger-than-average colonies.

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Chomvong et al. 199

Cellobiose consumption phenotypes of different CBP mutants were determined

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using S. cerevisiae D452-2 (MATα leu2 his3 ura3 can1), expressing pRS316- PTDH3-

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cdt1 F213L-TCYC1 and pRS315- PPGK1-cbp-TADH1. The strains were selected and

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maintained on oMM media supplied with appropriate CSM dropout mixtures. The In-

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Fusion HD Cloning Kit (Clontech) was used for all plasmid construction.

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The phenotypes were also evaluated under anaerobic conditions using oMM

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media provided with 8% cellobiose and 4% xylose with a starting OD600 of 20. The

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cultures were purged with nitrogen gas and sealed to maintain strict anaerobic

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conditions. Extracellular concentrations of cellobiose and xylose were determined by a

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Prominence HPLC (Shimadzu) equipped with Rezex RFP-FastAcid H 10 x 7.8 mm

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column. The column was eluted with 0.01 N of H2SO4 at a flow rate of 1 mL/min, 55°C.

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GX was quantified using an ICS-3000 Ion Chromatography System (Dionex), equipped

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with a CarboPac® PA200 Carbohydrate column. The column was eluted with a NaOAc

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gradient in 100 mM NaOH at a flow rate of 0.4 mL/min, 30°C.

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Protein purification Cellobiose phosphorylases fused with an N-terminal His6-tag were expressed in

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E. coli strain BL21 (DE3), induced by IPTG as described 3 and purified by HisTrap HP

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column (GE Healthcare) on the AKTA Explore FPLC system (GE Healthcare) according

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to the supplied protocol.

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Competition assay and kinetic parameters

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Chomvong et al. 221

10 nM of purified CBP was incubated with 5 mM inorganic phosphate and

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varying cellobiose concentrations at 30 °C in 20 mM MES, pH 6.0 with 0 or 10 mM

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xylose. G1P concentrations were continuously monitored as a readout using a G1P

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Colorimetry Assay Kit (Sigma-Aldrich). The G1P production rate was used for apparent

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Km and Vmax calculations by non-linear regression.

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4-O-ß-D-glucopyranosyl-D-xylose (GX) synthesis assay 200 nM of purified CBP was incubated with 10 mM G1P and 10 mM xylose or

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glucose in 20 mM MES, pH 6.0 at 30 °C for 1 hour. The reactions were stopped by

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adding 0.1 M NaOH, and concentrations of either GX or cellobiose were analyzed on

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the ICS-3000 Ion Chromatography System (Dionex), as described [3].

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CBP Homology and Modeling The SdCBP structure was predicted using Phyre2 Protein Homology/Analog

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Recognition Engine V 2.0 15. SdCBP homology to other CBP enzymes was determined

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using the phmmer algorithm (EMBL-EBI) 16, aligned using MUSCLE (EMBL-EBI) 17 and

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the sequence map was created using WebLogo (Berkeley) 18. Crystal structures of CBP

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in complexes with cellobiose 9 or glucose and sulfate 12 were used to model the Y47H

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mutation. Visualization, surface calculations and electrostatic calculations were carried

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out using PyMOL (Schrödinger) 19.

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Acknowledgements This work was supported by funding from Energy Biosciences Institute to JHDC.

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Chomvong et al. 244 245

Supporting Information. Supplementary figures and table

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(16) Finn, R. D., Clements, J., Arndt, W., Miller, B. L., Wheeler, T. J., Schreiber, F.,

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Bateman, A., and Eddy, S. R. (2015) HMMER web server: 2015 update. Nucleic Acids

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Res. 43, W30–8.

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(17) Edgar, R. C. (2004) MUSCLE: multiple sequence alignment with high accuracy and

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high throughput. Nucleic Acids Res. 32, 1792–1797.

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(18) Crooks, G. E., Hon, G., Chandonia, J.-M., and Brenner, S. E. (2004) WebLogo: a

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sequence logo generator. Genome Res. 14, 1188–1190.

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Figure Legends

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Figure 1. SdCBPCX9 and its use in aerobic growth and cellobiose consumption of

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a cellobiose-consuming S. cerevisiae strain. (A) The 7 mutations in the SdCBPCX9

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allele are labeled in red on a SdCBP structure predicted by Phyre2 15. (B) OD600 and

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cellobiose consumption profile of strains chromosomally expressing SdCBP-WT and

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SdCBPCX9 re-introduced into a clean strain background, in 2% cellobiose plus 1%

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xylose. Shown is a representative experiment.

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Figure 2. In vivo analysis of S. degradans CBP mutants. Extracellular

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concentrations of (A) cellobiose (B) xylose and (C) GX, using D452-2 strains expressing

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WT SdCBP or SdCBP mutants in anaerobic conditions, provided with 8% cellobiose 4%

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xylose. Experiments were carried out in biological triplicate, and include standard

320

deviations from the mean.

321 322

Figure 3. In vitro enzymology of S. degradans CBP mutants. (A) Percentage

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difference in kinetic parameters, in the presence or absence of 10 mM xylose, of

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purified WT SdCBP and SdCBP mutants. (B) Ratio of GX to cellobiose produced in the

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end-point reverse reaction assays using purified WT SdCBP or SdCBP mutants. All

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experiments in A and B were carried out in triplicate, and are shown with standard

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deviations from the mean.

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Chomvong et al. 329

Figure 4. Conservation of Y47H mutant phenotype in C. gilvus CBP. (A) Amino acid

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conservation among CBP homologs, near the protein N-terminus surrounding position

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47. (B) Anaerobic cellobiose consumption by strains expressing WT CgCBP or the

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Cgcbp Y47H mutant, provided with 8% cellobiose 4% xylose. The starting OD600 was

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20. (C) Percentage difference in kinetic parameters, in the presence or absence of 10

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mM xylose, of purified WT CgCBP or the Cgcbp Y47H mutant. (D) Ratio of GX to

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cellobiose produced in the end-point reverse reaction assays, using purified WT CgCBP

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or the Cgcbp Y47H mutant. In (B)-(D), experiments were carried out in triplicate, with

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standard deviations from the mean shown.

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Figure 5. In silico analysis of Y47H mutation in CBP structure. (A) Overview of

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Cellulomonas uda CBP structure in complex with cellobiose (denoted in blue). Tyrosine

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47 is shown in red. (B) Computational replacement of Y47H. (C) Surface view of the

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CBP structure with the Y47H mutation denoted in red. (D) Electrostatic map

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representation of the CBP structure. Blue represents positive electrostatic potential,

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whereas red represents negative potential. Green indicates the positions of Y47 at the

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dimer interface.

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Figure 1 138x238mm (300 x 300 DPI)



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Figure 2 249x83mm (300 x 300 DPI)


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Figure 3 113x174mm (300 x 300 DPI)



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Figure 4 245x119mm (300 x 300 DPI)


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Figure 5 139x137mm (300 x 300 DPI)


Relief of Xylose Binding to Cellobiose Phosphorylase by a Single

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