Integrating Structure, Bioinformatics, and Enzymology to Discover Function

Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 Å resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure. Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 Å resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure. The protein complement of both prokaryotes and eukaryotes remains largely uncharacterized. At least 30% of all proteins have no known biochemical function, and a larger percentage have sequence similarity to proteins of known biochemical activity (e.g. most predicted protein kinases) but for which the physiological role is unknown. The challenge in the post-genomic era is to define both the biochemical and physiological functions of all proteins as rapidly as possible.Structural proteomics, the large scale determination of protein structure, is expected to provide insight into the fundamental mechanisms by which a protein sequence adopts a defined three-dimensional structure. Most of the organized efforts in structural proteomics (Ref. 1Stevens R.C. Yokoyama S. Wilson I.A. Science. 2001; 294: 89-92Crossref PubMed protein for which is no known structural in the data at a of 30% sequence of is to define the of protein protein structure is often in the of sequence the of protein structures with of known proteins provide to biochemical of biochemical function from a protein structure with automated for structural of known function. The of are as with The of are used in the structural and have for detecting structural and for the for S. However, the of the from structural often a of and is by the to which the is to the an to the by which function is from structure, we have combined to we have a data of structural templates from the active of different of in in This the that the of the the and the of the catalytic and in of the protein are different PubMed the catalytic in is no similarity in structure. we have and used a panel of biochemical assays to the by the structural assays are often the is to the of the active and the we the of the combined and enzymatic of Escherichia coli a the for Structural the crystal structure of BioH with known we that BioH is a of the protein hydrolase and a catalytic screen with different hydrolase revealed that BioH carboxylesterase with a preference for short acyl chain and activity. The strategy used for BioH of novel, uncharacterized proteins and structures structural proteomics and of was by from coli The was as previously S. S. S. 2001; PubMed into the and of a of in which a the and a was from the The protein was and using as previously S. S. S. 2001; PubMed the of the BioH was in the coli in The was the as the protein for the of to the was by in of of protein to of and at in the with the with as a and in of the data and using used was a of the protein was in with was in using as a in in for by was a using an of and of the a using with a to the and and to of the protein. by and using the of The determined from the using with screening for enzyme using the activity was at using of as activity was using of and as PubMed activity as was by the of with PubMed activity was using as R.C. PubMed PubMed activity was determined using in at PubMed and activity was with as previously PubMed was the of the Structural at of the data at crystal protein. The crystal to the with a The data was using strategy at the and at a The was determined from the and the and with the 2001; data from the of the which had in of the data with the PubMed and of the data and was with PubMed and all of the data and with the of the of data and are in of data and of of of a of for the data of for the data the the the for the data the in a of BioH data was with the PubMed from to Å with using data at the the was with the PubMed The of the of the chain to and most of the to with a of The of the was and all of the using the PubMed This was the 1.7 Å data with of and the was and with the and was with PubMed the PubMed of The and are in and for BioH of of of all of from in a have in the BioH of the BioH crystal structure of of and The of the and to the protein as a of the The of the and of the of the of from the to the protein of was identified as of in and is a The of and have is a protein The of the large and of a by parallel and and both by and and into the and This the which is in is by the of the chain and is into the catalytic The and that a of The are by a and The is by and that the of catalytic and the of and the of and and Structural a of the of structural proteomics is to automated of protein structures to the of screen structures for catalytic function, we have a data of three-dimensional enzyme active structural in The BioH structure was data of using the PubMed This automated a of BioH to the catalytic triad of PubMed The BioH involved (Ser82, His235, and Asp207) the with a of Å for the This is the of Å used for from for The of the catalytic triad that BioH activity. the is of the identified PubMed which is for and of the His235, and the catalytic triad The are by the the BioH are by the and the chain The in the and is structure of BioH was with all known structures using as the PubMed The from the revealed structural to a large of proteins with a of enzymatic The with structural a an and and and and and a of BioH with a is in The sequence BioH and proteins from to and a specific catalytic function for of and have revealed to the that a catalytic triad in active of the coli BioH and of the catalytic the structure The of the is the for both the of the of and to the catalytic by a structural for the of the BioH catalytic The experimental an that from the chain of The of the and that the was to the of and with the of and the of the we the and BioH with with of to the and with similar of the protein with the at to and the at to in a was with of as the of the of BioH that the was and by both and the a the catalytic of for to of the catalytic and of the are with and with the from the of to biochemical was as but of are of and the of the and of and are with and and and the BioH protein was in the of which is known to with the catalytic in PubMed and a that BioH was The protein in the of that the to the of at and that the a in in the of was to enzymatic assays that hydrolase function and BioH carboxylesterase activity and of The enzyme and with a preference for short chain The of BioH determined for that BioH was most active with the for all was the with the of the and BioH was by of activity of with BioH and for all of the for coli BioH carboxylesterase activity with in a activity of BioH with acyl chain of protein with of an of the from at least with by BioH enzymatic for as a and as a and no enzymatic activity for as a as a and as data combined with in the that BioH a carboxylesterase in coli is known to at least proteins with carboxylesterase carboxylesterase S. PubMed PubMed PubMed and S. PubMed BioH no sequence similarity with BioH different with the with and for the chain and The specific activity of BioH for short was in the as for and at least as with BioH and activity with for but no activity with as a The of was for and for for the short chain from the that the catalytic of BioH is and is for with acyl chain of to could the in the of BioH the as a of the of the active are of acyl to BioH is to by and the active is large to short chain with similar for the This is with the that BioH for for BioH in and biotin is from by the and in a PubMed PubMed Escherichia coli and and the as is from S. PubMed using the and BioH proteins PubMed biochemical have The is in is in in to by and PubMed as and is from by S. PubMed S. PubMed to the of in coli using have S. PubMed but a Most a the of into with PubMed identified in BioH that are of and BioH was to from to and the coli protein function as an protein involved in The of a PubMed a role for BioH as a to a protein biochemical and structural data are with the of the BioH which that BioH from to PubMed and both and that BioH carboxylesterase and and that BioH use for we that the function of BioH is to and into and which are in the crystal structure but are could is that which is to function as a specific protein in the of PubMed with BioH and the of a to the BioH catalytic structures are for proteins of function. as for the structural data combined with in the the of an the in the to and biochemical function. However, as structural proteomics efforts an in the of protein structures for which is no of The of proteins that are in a specific of The three-dimensional structure of BioH was using automated for structural and with a of enzymatic This to rapidly BioH enzymatic activity and a enzyme in The and of combined the of structural in the by biochemical enzymatic functions to complement the structural The protein complement of both prokaryotes and eukaryotes remains largely uncharacterized. At least 30% of all proteins have no known biochemical function, and a larger percentage have sequence similarity to proteins of known biochemical activity (e.g. most predicted protein kinases) but for which the physiological role is unknown. The challenge in the post-genomic era is to define both the biochemical and physiological functions of all proteins as rapidly as Structural proteomics, the large scale determination of protein structure, is expected to provide insight into the fundamental mechanisms by which a protein sequence adopts a defined three-dimensional structure. Most of the organized efforts in structural proteomics (Ref. 1Stevens R.C. Yokoyama S. Wilson I.A. Science. 2001; 294: 89-92Crossref PubMed protein for which is no known structural in the data at a of 30% sequence of is to define the of protein protein structure is often in the of sequence the of protein structures with of known proteins provide to biochemical function. The of biochemical function from a protein structure with automated for structural of known function. The of are as with The of are used in the structural and have for detecting structural and for the for S. However, the of the from structural often a of and is by the to which the is to the an to the by which function is from structure, we have combined to we have a data of structural templates from the active of different of in in This the that the of the the and the of the catalytic and in of the protein are different PubMed the catalytic in is no similarity in structure. we have and used a panel of biochemical assays to the by the structural assays are often the is to the of the active and the Here we the of the combined and enzymatic of Escherichia coli a the for Structural the crystal structure of BioH with known we that BioH is a of the protein hydrolase and a catalytic screen with different hydrolase revealed that BioH carboxylesterase with a preference for short acyl chain and activity. The strategy used for BioH of novel, uncharacterized proteins and structures structural proteomics and of was by from coli The was as previously S. S. S. 2001; PubMed into the and of a of in which a the and a was from the The protein was and using as previously S. S. S. 2001; PubMed the of the BioH was in the coli in The was the as the protein for the of to the was by in of of protein to of and at in the with the with as a and in of the data and using used was a of the protein was in with was in using as a in in for by was a using an of and of the a using with a to the and and to of the protein. by and using the of The determined from the using with screening for enzyme using the activity was at using of as activity was using of and as PubMed activity as was by the of with PubMed activity was using as R.C. PubMed PubMed activity was determined using in at PubMed and activity was with as previously PubMed was the of the Structural at of the data at crystal protein. The crystal to the with a The data was using strategy at the and at a The was determined from the and the and with the 2001; data from the of the which had in of the data with the PubMed and of the data and was with PubMed and all of the data and with the of the of data and are in of data and of of of a of for the data of for the data the the the for the data the in a of BioH data was with the PubMed from to Å with using data at the the was with the PubMed The of the of the chain to and most of the to with a of The of the was and all of the using the PubMed This was the 1.7 Å data with of and the was and with the and was with PubMed the PubMed of The and are in and for BioH of of of all of from in a have in the BioH and of was by from coli The was as previously S. S. S. 2001; PubMed into the and of a of in which a the and a was from the The protein was and using as previously S. S. S. 2001; PubMed the of the BioH was in the coli in The was the as the protein for the of to the was by in of of protein to of and at in the with the with as a and in of the data and using used was a of the protein was in with was in using as a in in for by was a using an of and of the a using with a to the and and to of the protein. by and using the of The determined from the using with screening for enzyme using the activity was at using of as activity was using of and as PubMed activity as was by the of with PubMed activity was using as R.C. PubMed PubMed activity was determined using in at PubMed and activity was with as previously PubMed was the of the Structural at of the data at crystal protein. The crystal to the with a The data was using strategy at the and at a The was determined from the and the and with the 2001; data from the of the which had in of the data with the PubMed and of the data and was with PubMed and all of the data and with the of the of data and are in of BioH data was with the PubMed from to Å with using data at the the was with the PubMed The of the of the chain to and most of the to with a of The of the was and all of the using the PubMed This was the 1.7 Å data with of and the was and with the and was with PubMed the PubMed of The and are in have in the BioH of the BioH crystal structure of of and The of the and to the protein as a of the The of the and of the of the of from the to the protein of was identified as of in and is a The of and have is a protein The of the large and of a by parallel and and both by and and into the and This the which is in is by the of the chain and is into the catalytic The and that a of The are by a and The is by and that the of catalytic and the of and the of and and Structural a of the of structural proteomics is to automated of protein structures to the of screen structures for catalytic function, we have a data of three-dimensional enzyme active structural in The BioH structure was data of using the PubMed This automated a of BioH to the catalytic triad of PubMed The BioH involved (Ser82, His235, and Asp207) the with a of Å for the This is the of Å used for from for The of the catalytic triad that BioH activity. the is of the identified PubMed which is for and structure of BioH was with all known structures using as the PubMed The from the revealed structural to a large of proteins with a of enzymatic The with structural a an and and and and and a of BioH with a is in The sequence BioH and proteins from to and a specific catalytic function for of and have revealed to the that a catalytic triad in active of the coli BioH and of the catalytic the structure The of the is the for both the of the of and to the catalytic by a structural for the of the BioH catalytic The experimental an that from the chain of The of the and that the was to the of and with the of and the of the we the and BioH with with of to the and with similar of the protein with the at to and the at to in a was with of as the of the of BioH that the was and by both and the a the catalytic of for to of the catalytic and of the are with and with the from the of to biochemical was as but of are of and the of the and of and are with and and and the BioH protein was in the of which is known to with the catalytic in PubMed and a that BioH was The protein in the of that the to the of at and that the a in in the of was to enzymatic assays that hydrolase function and BioH carboxylesterase activity and of The enzyme and with a preference for short chain The of BioH determined for that BioH was most active with the for all was the with the of the and BioH was by of activity of with BioH and for all of the for coli BioH carboxylesterase activity with in a activity of BioH with acyl chain of protein with of an of the from at least with by BioH enzymatic for as a and as a and no enzymatic activity for as a as a and as data combined with in the that BioH a carboxylesterase in coli is known to at least proteins with carboxylesterase carboxylesterase S. PubMed PubMed PubMed and S. PubMed BioH no sequence similarity with BioH different with the with and for the chain and The specific activity of BioH for short was in the as for and at least as with BioH and activity with for but no activity with as a The of was for and for for the short chain from the that the catalytic of BioH is and is for with acyl chain of to could the in the of BioH the as a of the of the active are of acyl to BioH is to by and the active is large to short chain with similar for the This is with the that BioH for for BioH in and biotin is from by the and in a PubMed PubMed Escherichia coli and and the as is from S. PubMed using the and BioH proteins PubMed biochemical have The is in is in in to by and PubMed as and is from by S. PubMed S. PubMed to the of in coli using have S. PubMed but a Most a the of into with PubMed identified in BioH that are of and BioH was to from to and the coli protein function as an protein involved in The of a PubMed a role for BioH as a to a protein biochemical and structural data are with the of the BioH which that BioH from to PubMed and both and that BioH carboxylesterase and and that BioH use for we that the function of BioH is to and into and which are in the crystal structure but are could is that which is to function as a specific protein in the of PubMed with BioH and the of a to the BioH catalytic structures are for proteins of function. as for the structural data combined with in the the of an the in the to and biochemical function. However, as structural proteomics efforts an in the of protein structures for which is no of The of proteins that are in a specific of The three-dimensional structure of BioH was using automated for structural and with a of enzymatic This to rapidly BioH enzymatic activity and a enzyme in The and of combined the of structural in the by biochemical enzymatic functions to complement the structural The BioH of the BioH crystal structure of of and The of the and to the protein as a of the The of the and of the of the of from the to the protein of was identified as of in and is a The of and have BioH is a protein The of the large and of a by parallel and and both by and and into the and This the which is in is by the of the chain and is into the catalytic The and that a of The are by a and The is by and that the of catalytic and the of and the of and and Structural a of the of structural proteomics is to automated of protein structures to the of screen structures for catalytic function, we have a data of three-dimensional enzyme active structural in The BioH structure was data of using the PubMed This automated a of BioH to the catalytic triad of PubMed The BioH involved (Ser82, His235, and Asp207) the with a of Å for the This is the of Å used for from for The of the catalytic triad that BioH activity. the is of the identified PubMed which is for and The structure of BioH was with all known structures using as the PubMed The from the revealed structural to a large of proteins with a of enzymatic The with structural a an and and and and and a of BioH with a is in The sequence BioH and proteins from to and a specific catalytic function for of and have revealed to the that a catalytic triad in active sites. by a structural for the of the BioH catalytic The experimental an that from the chain of The of the and that the was to the of and with the of and the of the we the and BioH with with of to the and with similar of the protein with the at to and the at to in a was with of as the of the of BioH that the was and by both and the a the catalytic of for to and and the BioH protein was in the of which is known to with the catalytic in PubMed and a that BioH was The protein in the of that the to the of at and that the BioH a in in the of was to enzymatic assays that hydrolase function and BioH carboxylesterase activity and of The enzyme and with a preference for short chain The of BioH determined for that BioH was most active with the for all was the with the of the and BioH was by of activity of with BioH and for all of the BioH enzymatic for as a and as a and no enzymatic activity for as a as a and as data combined with in the that BioH a carboxylesterase in coli is known to at least proteins with carboxylesterase carboxylesterase S. PubMed PubMed PubMed and S. PubMed BioH no sequence similarity with BioH different with the with and for the chain and The specific activity of BioH for short was in the as for and at least as with BioH and activity with for but no activity with as a The of was for and for The for the short chain from the that the catalytic of BioH is and is for with acyl chain of to could the in the of BioH the as a of the of the active are of acyl to BioH is to by and the active is large to short chain with similar for the This is with the that BioH for for BioH in and biotin is from by the and in a PubMed PubMed Escherichia coli and and the as is from S. PubMed using the and BioH proteins PubMed biochemical have The is in is in in to by and PubMed as and is from by S. PubMed S. PubMed to the of in coli using have S. PubMed but a Most a the of into with PubMed identified in BioH that are of and BioH was to from to and the coli protein function as an protein involved in The of a PubMed a role for BioH as a to a protein biochemical and structural data are with the of the BioH which that BioH from to PubMed and both and that BioH carboxylesterase and and that BioH use for we that the function of BioH is to and into and which are in the crystal structure but are could is that which is to function as a specific protein in the of PubMed with BioH and the of a to the BioH catalytic structures are for proteins of function. as for the structural data combined with in the the of an the in the to and biochemical function. However, as structural proteomics efforts an in the of protein structures for which is no of The of proteins that are in a specific of The three-dimensional structure of BioH was using automated for structural and with a of enzymatic This to rapidly BioH enzymatic activity and a enzyme in The and of combined the of structural in the by biochemical enzymatic functions to complement the structural all of the Structural at and of the for Structural for in and for

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