A structural explanation for the mechanism and specificity of plant branching enzymes I and IIb

Abstract

Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme’s surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs. Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme’s surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs. Starch, the primary energy storage molecule of plants, is a complex biomaterial built from large α-1,4- and α-1,6-branched glucose polymers packed into a well-ordered granule, with amorphous and semicrystalline layers throughout the granule (1Tetlow I.J. Emes M.J. A review of starch-branching enzymes and their role in amylopectin biosynthesis.IUBMB Life. 2014; 66: 546-558Crossref PubMed Scopus (98) Google Scholar, 2Pérez S. Bertoft E. The molecular structures of starch components and their contribution to the architecture of starch granules: A comprehensive review.Starch - Stärke. 2010; 62: 389-420Crossref Scopus (864) Google Scholar). Two types of polymer make up these granules: Amylose, a smaller polymer (20–30% of starch weight, with a degree of polymerization (DP) of 200–500) almost devoid of σ-1,6 branches (3Curá J.A. Jansson P.-E. Krisman C.R. Amylose is not strictly linear.Starch - Stärke. 1995; 47: 207-209Crossref Scopus (34) Google Scholar) (with only 5–20 branches per molecule); and Amylopectin, a much larger polymer (65–85% starch weight, with average DP 11–15 for short chains, average DP 43–50 for long chains) with approximately 5 to 6% branching (4Buleon A. Colonna P. Planchot V. Ball S. Starch granules: Structure and biosynthesis.Int. J. Biol. Macromol. 1998; 23: 85-112Crossref PubMed Scopus (1436) Google Scholar, 5Nakamura Y. (Bio)Chemical and Structural Properties of Starch and Glycogen.Starch, Metabolism and Structure. Springer, Berlin, Heidelberg2015Google Scholar). The starch granule’s size, density, and fine structure are key to its function as the primary energy storage unit for all plants, leading to more or less bioavailability and robustness, depending on the plant’s energy requirements (6Blennow A. Engelsen S.B. Helix-breaking news: Fighting crystalline starch energy deposits in the cell.Trends Plant Sci. 2010; 15: 236-240Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The starch granule is biosynthesized by the interplay of several enzymes, including ADP-glucose pyrophosphorylase, which makes the ADP-glucose monomer building block, starch synthases, which convert ADP-glucose to α-1,4-linked glucan polymers, branching enzymes, which cleave α-1,4-linkages and transfer the resulting cleaved glucan to α-1,6 positions to form branches, debranching enzymes, which remove inappropriately placed branches, and kinases that phosphorylate the resulting polymer (7Lü B. Guo Z. Liang J. Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms.Sci. China C Life Sci. 2008; 51: 863-871Crossref PubMed Scopus (13) Google Scholar). These enzymes all work in concert to build the starch granule. Most of these enzymes have several isoforms, each with unique roles to play in constructing the granule (8Rahman S. Comparison of starch-branching enzyme genes reveals evolutionary relationships among isoforms. Characterization of a gene for starch-branching enzyme IIa from the wheat D genome donor Aegilops tauschii.Plant Physiol. 2001; 125: 1314-1324Crossref PubMed Scopus (92) Google Scholar, 9Denyer K. Sidebottom C. Hylton C.M. Smith A.M. Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos.Plant J. 1993; 4: 191-198Crossref PubMed Scopus (109) Google Scholar). Branching enzyme (BE) has at least two and often three isoforms in most plants, BEI, BEIIa, and BEIIb, that differ in both substrate and product specificity (1Tetlow I.J. Emes M.J. A review of starch-branching enzymes and their role in amylopectin biosynthesis.IUBMB Life. 2014; 66: 546-558Crossref PubMed Scopus (98) Google Scholar, 10Mizuno K. Kobayashi E. Tachibana M. Kawasaki T. Fujimura T. Funane K. Kobayashi M. Baba T. Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds.Plant Cell Physiol. 2001; 42: 349-357Crossref PubMed Scopus (58) Google Scholar, 11Li C. Gilbert R.G. Progress in controlling starch structure by modifying starch-branching enzymes.Planta. 2016; 243: 13-22Crossref PubMed Scopus (26) Google Scholar, 12Wu A.C. Morell M.K. Gilbert R.G. A parameterized model of amylopectin synthesis provides key insights into the synthesis of granular starch.PLoS One. 2013; 8e65768Google Scholar). While BEI isoforms prefer amylose as a substrate and tend to transfer longer chains of 11 or more units, BEII isoforms favor amylopectin as a substrate and tend to transfer shorter chains of 6 to 7 glucose units, with BEIIa transferring a broader range of glucans while BEIIb isoforms transfer almost exclusively chains of 6 to 7 glucose units (10Mizuno K. Kobayashi E. Tachibana M. Kawasaki T. Fujimura T. Funane K. Kobayashi M. Baba T. Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds.Plant Cell Physiol. 2001; 42: 349-357Crossref PubMed Scopus (58) Google Scholar, 11Li C. Gilbert R.G. Progress in controlling starch structure by modifying starch-branching enzymes.Planta. 2016; 243: 13-22Crossref PubMed Scopus (26) Google Scholar, 12Wu A.C. Morell M.K. Gilbert R.G. A parameterized model of amylopectin synthesis provides key insights into the synthesis of granular starch.PLoS One. 2013; 8e65768Google Scholar, 13Nakamura Y. Utsumi Y. Sawada T. Aihara S. Utsumi C. Yoshida M. Kitamura S. Characterization of the reactions of starch branching enzymes from rice endosperm.Plant Cell Physiol. 2010; 51: 776-794Crossref PubMed Scopus (102) Google Scholar). They also differ in localization, with BEI and BEIIb almost exclusively found in the amyloplast. BEIIa is expressed throughout the plant, especially in the leaf, where it is exclusively responsible for branching the rapidly created and degraded starch (14Sun C. Sathish P. Ahlandsberg S. Jansson C. The two genes encoding starch-branching enzymes IIa and IIb are differentially expressed in barley.Plant Physiol. 1998; 118: 37-49Crossref PubMed Scopus (89) Google Scholar, 15Hamada S. Ito H. Hiraga S. Inagaki K. Nozaki K. Isono N. Yoshimoto Y. Takeda Y. Matsui H. Differential characteristics and subcellular localization of two starch-branching enzyme isoforms encoded by a single gene in Phaseolus vulgaris L.J. Biol. Chem. 2002; 277: 16538-16546Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). We and others have been working to elaborate the molecular details that give rise to the unique specificity of both glycogen and starch branching enzymes (16Abad M.C. Binderup K. Rios-Steiner J. Arni R.K. Preiss J. Geiger J.H. The X-ray crystallographic structure of Escherichia coli branching enzyme.J. Biol. Chem. 2002; 277: 42164-42170Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 17Feng L. Fawaz S. Gilbert L. J. Geiger J.H. structures of Escherichia coli branching enzyme in complex with PubMed Scopus (34) Google Scholar, L. Fawaz S. M. Geiger J.H. structures of Escherichia coli branching enzyme in complex with D Biol. 2016; PubMed Scopus Google Scholar, A. T. M. E. A. M. A. Structural of glycogen branching enzyme and by PubMed Scopus Google Scholar, Z. C. C. Z. L. Y. Z. and of an branching enzyme from PubMed Scopus Google Scholar, K. S. S. P. K. structure of glycogen branching of in substrate specificity and Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. Ball N. E. substrate in the structure of branching enzyme a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. T. Y. M. structure of the rice branching enzyme I (BEI) in complex with PubMed Scopus Google Scholar). we the structure of rice BEI we the acceptor chain binding site on the surface of the enzyme for the Mutagenesis confirm the importance of this binding site for activity while also that the site is not the donor chain binding site. Comparison of the structure with the Cyanothece BE structure us to identify the likely donor chain binding site M. C. Ball N. E. substrate in the structure of branching enzyme a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Mutagenesis of two loops in the of this donor chain binding site the activity and specificity of to to that of the location of the donor chain binding site. these results give the most complete picture to the mechanism of BE and identify the for the activity of the isoforms. These insights the and of BEs to their activities much more leading to more and of this most critical The structures of both and were K. J. T. Y. M. structure of the rice branching enzyme I (BEI) in complex with PubMed Scopus Google Scholar). binding sites were all from the active site. In the a form identified and the structure by these form a that by on its for activity and chain specificity assays this to to that of the two to A and the sites identified in the structure K. J. T. Y. M. structure of the rice branching enzyme I (BEI) in complex with PubMed Scopus Google Scholar). glucose units are in site and the between glucan and are to that in the structure The and of the are to that in an amylose single The molecule a binding site not identified in BE and has all glucose units at a on the from the active site and the active site the of the The glucan a with glucose units per to chain of a glycogen A. M. H. N. C. structure of A from of single 42: Scopus Google it from this as it the active site. The surface of is binding the with and that to the glucan from the surface and with the glucose units that the surface The that interact with in this binding site are in plant and with 11 of the that interact with in all plant and BEs is in BEs for this this binding site to to the BEs and from the enzymes, including the enzymes with BEs as Cyanothece E. of enzymes among Life Sci. 2016; PubMed Scopus (27) Google of binding sites and between and and The two structures were The surface and glucans of the structure and the glucans of the structure are from site on a model of an amylose single from 11 of the amylose single model on the structure of K. T. N. Smith S. at X-ray structure of a with glucose Sci. S. A. PubMed Scopus Google binding site between and site and the from are by for for from site on of a glycogen or from the and for plant and and Escherichia coli The of that in site is that or are to are When the structures of BE1 are with BE1 structures with and structural is from the between and is or found in two in the BEI binding this to a not in the make with the to the in the structure with the glucose found to the active site and as a into the active site. large are in the binding site all three structures are The of the and the to the active site that the binding site play a role in the of the enzyme. by and transfer chain specificity the activity transfer chain specificity for this enzyme is a and it to The of the transfer chain specificity is a critical the specificity is by the that BE is branching a in longer more chains and longer chains. The for this is that the longer chains by are also substrates for transfers to the branches that are long to substrates of the enzyme. enzymes with activity with enzymes that have activity by the in the by the transfer chain specificity for each only the of the at the activities of to the unit as the of the enzyme to the of the complex by per The activity on amylose substrate is which is to T. K. A. H. H. T. M. The of rice branching enzyme I (BEI) on PubMed Scopus Google Scholar). several in the binding site and of of these the chain specificity and We also identified a large of 11 in the to found in all BEII enzymes (10Mizuno K. Kobayashi E. Tachibana M. Kawasaki T. Fujimura T. Funane K. Kobayashi M. Baba T. Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds.Plant Cell Physiol. 2001; 42: 349-357Crossref PubMed Scopus (58) Google Scholar) is to the binding and in this make with that it an role in between BEI and BEII isoforms, an found in to the to in BEI a of activity in chain specificity identified of the most in BEI BEII activity is the of BEII enzymes for the transfer of shorter chains to BEI enzymes (10Mizuno K. Kobayashi E. Tachibana M. Kawasaki T. Fujimura T. Funane K. Kobayashi M. Baba T. Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds.Plant Cell Physiol. 2001; 42: 349-357Crossref PubMed Scopus (58) Google Scholar, A. Y. N. H. and of the of in rice endosperm.Plant Physiol. 2001; PubMed Scopus Google activities of and several key activities on are to the of activity of at is to the activity while for only the is the of activity of at is to the activity while for only the is the of activity of at is to the activity while for only the is the of activity of at is to the activity while for only the is the of activity of at is to the activity while for only the is the of activity of at is to the activity while for only the is chain chain chain chain chain and is as The activities on are to the of activity of at is to the activity while for only the is in a is as A to found to the to also between BEI and BEII enzymes When both this and the to were in BEI with the found in were in the the activity amylose as substrate the chain specificity of BEI from a for longer glucose chains to an almost for chains of 6 to 7 glucose units C and to that for We conclude that these two loops work to the chain specificity in and likely plant branching The unique function and specificity of branching enzymes, their role in and modifying their transfer chain depending on or and their to chains, make enzymes, the specificity for substrate and product of most a of branching enzyme structures are (16Abad M.C. Binderup K. Rios-Steiner J. Arni R.K. Preiss J. Geiger J.H. The X-ray crystallographic structure of Escherichia coli branching enzyme.J. Biol. Chem. 2002; 277: 42164-42170Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, A. T. M. E. A. M. A. Structural of glycogen branching enzyme and by PubMed Scopus Google Scholar, K. S. S. P. K. structure of glycogen branching of in substrate specificity and Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. T. Y. M. structure of the rice branching enzyme I (BEI) in complex with PubMed Scopus Google Scholar, J. K. T. H. T. A. H. T. Y. M. structure of the branching enzyme (BEI) from sativa with for and substrate PubMed Scopus Google of which are to the structural details that give rise to the unique characteristics of BEs mysterious. is the to at to a BE and a binding surface at a from the active and not the active site. confirm the importance of the binding surface for the activity of the enzyme, and the with are in BEs. this to the that the binding surface a of the donor or acceptor chain binding site. the from the active activity of we that the donor chain binding this can involved in between the donor chain and the enzyme only is transferring chains longer glucose The that the of chains smaller 11 glucose units for almost of all chains is with only In the that of the to this binding surface in in transfer chain length also its in donor chain binding L. Fawaz S. Gilbert L. J. Geiger J.H. structures of Escherichia coli branching enzyme in complex with PubMed Scopus (34) Google Scholar, K. S. S. P. K. structure of glycogen branching of in substrate specificity and Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. Ball N. E. substrate in the structure of branching enzyme a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a structure of Cyanothece BE M. C. Ball N. E. substrate in the structure of branching enzyme a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) for the a donor chain in the active site of a of the that the donor chain binding in the Cyanothece BE structure are in and including that all BEs a donor chain binding surface. The binding surface a from that of the surface to the that the binding surface of the acceptor chain binding site. the Cyanothece BE structure as a an into the donor chain binding surface of the donor chain between the to and to in a that the donor binding surface and the the is between the donor and acceptor chain binding are the to is between the donor and acceptor chain binding is the that is in BEIIb isoforms. of the with that of the activity of the enzyme and in a in chain specificity shorter chains, more to that of both this and the to chain into an in its product specificity. which is the isoform that transfer of the chains of of the isoforms chains of to is to an enzyme, that transfers almost exclusively only the and chains. these results the of plant BEs that is responsible for the specificity of the enzyme for the the to the of the A BEI and BEIIb the in chain transfer with the importance of this in the activity in and T. Preiss J. of enzymes of branching enzymes I and and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the to the that which of the enzyme were responsible for this specificity. on the of the donor chain binding site that the two loops the of the donor where to to play a role in chain specificity. to confirm that the donor chain is in to that of Cyanothece it likely that all BEs a donor chain binding surface. it that loops on both of the donor chain are for controlling donor chain We that the longer found in BEII enzymes reaches the donor chain binding with the to and the of an or donor chain to for shorter donor chains. is to that a in Cyanothece BE the of the with the of the glucose and likely provides of the specificity for shorter glucan chains in the Cyanothece enzyme. is not in enzymes, of which have chain from that of Cyanothece The of the to both donor and acceptor chains and the that in this make with the acceptor chain in the structure the that is between and sites that binding of not the binding of the in the active site. also that between donor and acceptor chains both are as for B. H. E. Y. Y. structure of for binding of in the active Biol. PubMed Scopus Google Scholar). these results that between donor and acceptor chains for acceptor chain binding within the active site. a acceptor chain binding from the donor chain from the active site. the with the surface of the enzyme, not into the active site. from the structure the and of the acceptor glucan of the occur in the of the it to binding of a in this binding site. while the of the to the active site are at least in the binding with with the of the from the surface and are not by that while the positions to have a the positions to a the binding of an that a on a within to 6 glucan units of the acceptor from branches together, while with an acceptor chain at a more 6 units from the acceptor with demonstrating with acceptor chains T. Y. T. A. E. N. T. S. M. C. Ball S. of characteristics of glucan branching enzymes and the fine structure of from 2014; PubMed Scopus Google Scholar). In with the insights from the Cyanothece BE structure and the structure with the results that the responsible for controlling donor chain specificity in plant isoforms, an picture of the critical branching in the biosynthesis of the starch granule to rice BEI gene from the of in of J. the of the gene by from the complete found on the the The and the were from and and were from Amylose substrate in assays from Amylose from were by for more The gene (with into an and of the gene of rise to the all and activity were with all the activity assays and chain length were with the and The of not from the from the the In to cleave the a site The at the were and as of the on the were in the The were at to and the with at were at at for 6 at The at for at The with and and and The were for all activity and chain length specificity for by the to cleaved with and for at leading to a by at its a almost to that found to have activity to that of The by from in a of and were this and were by in an The for are in The were to in the to the a of in the at and the to The BEI that not or in the in the polymer to these and the in and in at A of dodecaose in the and were in the polymer for which the were The were in of the in to and in The structure of with by molecular the rice structure as a The to complete to and to an of and of The X-ray were from a single at the The were The structure The structure molecule in the unit and to with unit a and X-ray structure and were in the as and structure rice branching enzyme per in for of allowed in a The the in of complex at Preiss J. of from developing L. Scopus Google Scholar). the amylose substrate is by the enzyme, the of the complex unit of activity is as a in of per at at and is in by of and of in and it for assays to Amylose by of amylose in and In to the amylose the by for Amylose working by amylose and with the to by at for 5 to amylose working at In of enzymes to the amylose working resulting in a enzyme of 5 and were into at for 5 and at and for were on the for active and the for less active and to the the chain amylose by the enzymes as in activity and the product of branching by The with to the of the chain length specificity in the as activity reactions were by the of complex at of its were from the of activity the in to the branching enzyme, the to to by The debranching in by units of from The debranching by and at for 5 to remove the from the a The resulting were in and and as length by by V. of from and 2013; PubMed Scopus Google Scholar). The to the rice BE I structure have been in the and The that have of with the of this by the and crystallographic were at the an of for the of of by by the of the by the the and the of the for The is the of the and not the of the of H. N. and J. H. H. N. and J. H. H. N. and J. H. J. H. H. N. N. K. S. Z. A. and J. H. H. N. N. S. and J. H. J. H. J. H. H. N. and J. H. H. N. and J. H. H. N. and J. H. H. N. and J. H. H. N. and J. H. and with and

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