生物素（維生素H）是由植物和大多數原核生物合成，是所有生物體所必需的，生物素通常作為參與二氧化碳轉移的酵素輔助因子。生物素蛋白連接酶（Biotin protein ligase；BPL），也稱為高羧酸酶合成酶(Holocarboxylase synthetase；HCS)或BirA，是負責將生物素共價連接到受質蛋白質上的酶，此蛋白質修飾雖然普遍存在所有生物，且是必要，卻是屬於比較罕見的轉譯後修飾，通常一個物種內只有1～5個蛋白質受質。BPL將生物素鍵結到受質蛋白質上特定的賴胺酸，這個賴胺酸通常位於一段保守序列AMKM中，因此生物素化具有高度專一性。Arc1p是一個Saccharomyces cerevisiae酵母菌非專一性tRNA結合蛋白質，在細胞質內與glutamyl-tRNA synthetase及methionyl-tRNA synthetase形成三聚體，除能增強此二酵素的胺醯化活性，也能調控他們的細胞內分布。有趣的是，Arc1p缺少生物素化必要的保守序列AMKM，但它卻可以被BPL1生物素化，且多數酵母菌種的BPL1無法生物素化其Arc1p，最近的研究報告發現，Arc1p包含一個獨特的生物素化位點SSKD。 本研究具體目標是想找出酵母菌BPL及其受質的專一性部位，藉由Protein Data Bank中的晶體結構模型得知，AMKM及SSKD的結構差異頗大，進一步利用點突變實驗發現， SSKD附近的胺基酸序列對於生物素化很重要，改變這些胺基酸會明顯降低其生物素化的程度，將Arc1p的SSKD突變成AMKM後，Arc1p就不再能被生物素化。另外，透過序列比對，我們發現在S. cerevisiae酵母菌BPL1的 C端有一些獨特的嵌入胜肽，這些獨特的胜肽序列不存在多數酵母菌BPL1中，然而刪除這些獨特的胜肽序列並不會影響其生物素化Arc1p的能力。除此之外，透過不同酵母菌種之間交換BPL1片段，也無法準確定位酵素專一性區域，目前仍有待進一步的研究去解開酵母菌BPL1的專一性謎團。

Biotin (vitamin H), an essential coenzyme synthesized by plants and most prokaryotes, is required by all organisms. Biotin serves as a cofactor for enzymes that participates in carbon dioxide transfer. Biotin protein ligase (BPL), also known as holocarboxylase synthetase (HCS) or BirA, is the enzyme responsible for attaching biotin to proteins. Although this modification is ubiquitous and necessary for all organisms, a relatively rare post-translational modification. Only one to five protein acceptors in every single species. BPL catalyzes the attachment of biotin to a specific lysine residue located within a conserved sequence AMKM. Arc1p is a non-specific tRNA-binding protein of Saccharomyces cerevisiae. It forms a ternary complex in the cytoplasm with glutamyl-tRNA synthetase and methionyl-tRNA synthetase, and enhances the aminoacylation activity of these two enzymes. In addition, Arc1p also regulates the intracellular distributions of these two enzymes. While Arc1p lacks an AMKM motif, it can be specifically biotinylated by BPL1. In contrast, BPL1s of most yeast species cannot biotinylate their own Arc1p homologues. Recent studies have found that Arc1p contains a unique biotinylation site, SSKD. The aim of this project is to map the substrate-specific site of yeast BPL. According to the crystal structure models in Protein Data Bank, the structures of AMKM and SSKD are quite different. Site-directed Mutagenesis further showed that amino acid sequences flanking SSKD are important for biotinylation, and altering these amino acids significantly reduces the biotinylation level. Arc1p could not be biotinylated when its SSKD was mutated to AMKM. Also, we found that there are some unique embedded peptides in the C-terminal domain of S. cerevisiae BPL1. These unique peptides are absent ftrom most yeast BPL1 homologues. However, deleting these unique peptide sequences did not affect its ability to biotinylate Arc1p. In addition, exchange of N and C domains between BPL1s of different yeast species failed to locate the substrate-specific region accurately. Further research is underway to map the substrate-specific site of yeast BPL1.

Bagautdinov B, Kuroishi C, Sugahara M, Kunishima N. 2005. Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation. J Mol Biol 353:322-333.

Bockman MR, Kalinda AS, Petrelli R, De la Mora-Rey T, Tiwari D, Liu F, Dawadi S, Nandakumar M, Rhee KY, Schnappinger D, et al. 2015. Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors. J Med Chem 58:7349-7369.

Chang CY, Chang CP, Chakraborty S, Wang SW, Tseng YK, Wang CC. 2016. Modulating the Structure and Function of an Aminoacyl-tRNA Synthetase Cofactor by Biotinylation. J Biol Chem 291:17102-17111.

Chang KJ, Wang CC. 2004. Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae. J Biol Chem 279:13778-13785.

Chapman-Smith A, Cronan JE, Jr. 1999a. The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity. Trends Biochem Sci 24:359-363.

Chapman-Smith A, Cronan JE, Jr. 1999b. Molecular biology of biotin attachment to proteins. J Nutr 129:477S-484S.

Cole H, Reynolds TR, Lockyer JM, Buck GA, Denson T, Spence JE, Hymes J, Wolf B. 1994. Human serum biotinidase. cDNA cloning, sequence, and characterization. J Biol Chem 269:6566-6570.

Cronan JE, Jr. 1990. Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J Biol Chem 265:10327-10333.

Frechin M, Kern D, Martin RP, Becker HD, Senger B. 2010. Arc1p: anchoring, routing, coordinating. FEBS Lett 584:427-433.

Hassan YI, Moriyama H, Olsen LJ, Bi X, Zempleni J. 2009. N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition. Mol Genet Metab 96:183-188.

Healy S, McDonald MK, Wu X, Yue WW, Kochan G, Oppermann U, Gravel RA. 2010. Structural impact of human and Escherichia coli biotin carboxyl carrier proteins on biotin attachment. Biochemistry 49:4687-4694.
Karanasios E, Simader H, Panayotou G, Suck D, Simos G. 2007. Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly. J Mol Biol 374:1077-1090.

Kim HS, Hoja U, Stolz J, Sauer G, Schweizer E. 2004. Identification of the tRNA-binding protein Arc1p as a novel target of in vivo biotinylation in Saccharomyces cerevisiae. J Biol Chem 279:42445-42452.

Kuroishi T. 2015. Regulation of immunological and inflammatory functions by biotin. Can J Physiol Pharmacol 93:1091-1096.

Lee CK, Cheong C, Jeon YH. 2010. Substrate recognition characteristics of human holocarboxylase synthetase for biotin ligation. Biochem Biophys Res Commun 391:455-460.

Lee CK, Cheong HK, Ryu KS, Lee JI, Lee W, Jeon YH, Cheong C. 2008. Biotinoyl domain of human acetyl-CoA carboxylase: Structural insights into the carboxyl transfer mechanism. Proteins 72:613-624.

Leon-Del-Rio A, Gravel RA. 1994. Sequence requirements for the biotinylation of carboxyl-terminal fragments of human propionyl-CoA carboxylase alpha subunit expressed in Escherichia coli. J Biol Chem 269:22964-22968.

Li Y, Hassan YI, Moriyama H, Zempleni J. 2013. Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events. J Nutr Biochem 24:1446-1452.

Ma Q, Akhter Y, Wilmanns M, Ehebauer MT. 2014. Active site conformational changes upon reaction intermediate biotinyl-5'-AMP binding in biotin protein ligase from Mycobacterium tuberculosis. Protein Sci 23:932-939.

Manthey KC, Griffin JB, Zempleni J. 2002. Biotin supply affects expression of biotin transporters, biotinylation of carboxylases and metabolism of interleukin-2 in Jurkat cells. J Nutr 132:887-892.

O'Callaghan C A, Byford MF, Wyer JR, Willcox BE, Jakobsen BK, McMichael AJ, Bell JI. 1999. BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation. Anal Biochem 266:9-15.

Pendini NR, Bailey LM, Booker GW, Wilce MC, Wallace JC, Polyak SW. 2008. Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay. Arch Biochem Biophys 479:163-169.
Pendini NR, Yap MY, Traore DA, Polyak SW, Cowieson NP, Abell A, Booker GW, Wallace JC, Wilce JA, Wilce MC. 2013. Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target. Protein Sci 22:762-773.

Polyak SW, Chapman-Smith A, Brautigan PJ, Wallace JC. 1999. Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity. J Biol Chem 274:32847-32854.

Procter M, Wolf B, Crockett DK, Mao R. 2013. The Biotinidase Gene Variants Registry: A Paradigm Public Database. G3 (Bethesda) 3:727-731.

Procter M, Wolf B, Mao R. 2016. Forty-eight novel mutations causing biotinidase deficiency. Mol Genet Metab 117:369-372.

Ripmaster TL, Shiba K, Schimmel P. 1995. Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen. Proc Natl Acad Sci U S A 92:4932-4936.

Sikorski RS, Hieter P. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27.

Simader H, Hothorn M, Kohler C, Basquin J, Simos G, Suck D. 2006. Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes. Nucleic Acids Res 34:3968-3979.

Sternicki LM, Wegener KL, Bruning JB, Booker GW, Polyak SW. 2017. Mechanisms Governing Precise Protein Biotinylation. Trends Biochem Sci 42:383-394.

Tron CM, McNae IW, Nutley M, Clarke DJ, Cooper A, Walkinshaw MD, Baxter RL, Campopiano DJ. 2009. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity. J Mol Biol 387:129-146.

Val DL, Chapman-Smith A, Walker ME, Cronan JE, Jr., Wallace JC. 1995. Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation. Biochem J 312 ( Pt 3):817-825.

Wilson KP, Shewchuk LM, Brennan RG, Otsuka AJ, Matthews BW. 1992. Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc Natl Acad Sci U S A 89:9257-9261.

Zempleni J, Liu D, Camara DT, Cordonier EL. 2014. Novel roles of holocarboxylase synthetase in gene regulation and intermediary metabolism. Nutr Rev 72:369-376.