A Newly Determined Member of themeso-Diaminopimelate Dehydrogenase Family with a Broad Substrate Spectrum.
meso-Diaminopimelate dehydrogenase (meso-DAPDH) fromSymbiobacterium thermophilum(StDAPDH) is the first member of themeso-DAPDH family known to catalyze the asymmetric reductive amination of 2-keto acids to produce d-amino acids. It is important to understand the catalytic mechanisms of StDAPDH and other enzymes in this family. In this study, based on an evolutionary analysis and examination of catalytic activity, themeso-DAPDH enzymes can be divided into two types. Type I showed highly preferable activity towardmeso-diaminopimelate (meso-DAP), and type II exhibited obviously reversible amination activity with a broad substrate spectrum. StDAPDH belongs to type II. A quaternary structure analysis revealed that insertions/deletions (indels) and a loss of quaternary structure resulted in divergence among members of themeso-DAPDH family. A structure alignment of StDAPDH with a representative of type I, themeso-DAPDH fromCorynebacterium glutamicum(CgDAPDH), indicated that they had the same folding. Based on sequence and conservation analyses, two amino acid residues of StDAPDH, R35 and R71, were found to be highly conserved within type II while also distinct from each other between the subtypes. Site mutagenesis studies identified R71 as a substrate preference-related residue of StDAPDH, which may serve as an indicator of the amination preference of type II. These results deepen the present understanding of themeso-DAPDH family and provide a solid foundation for the discovery and engineering ofmeso-DAPDH for d-amino acid biosynthesis.IMPORTANCEThe l-form of amino acids is typically more abundant than the d-form. However, the d-form has many important pharmaceutical applications.meso-Diaminopimelate dehydrogenase (meso-DAPDH) fromSymbiobacterium thermophilum(StDAPDH) was the first member ofmeso-DAPDH known to catalyze the amination of 2-keto acids to produce d-amino acids. Accordingly, we analyzed the evolution ofmeso-DAPDH proteins and found that they form two groups, i.e., type I proteins, which show high preference towardmeso-diaminopimelate (meso-DAP), and type II proteins, which show a broad substrate spectrum. We examined the differences in sequence, ternary structure, and quaternary structure to determine the mechanisms underlying the functional differences between the type I and type II lineages. These results will facilitate the identification of additionalmeso-DAPDHs and may provide guidance to protein engineering studies for d-amino acid biosynthesis.
School of Life Science, Shandong University of Technology, Zibo, People's Republic of China gaoxz@sdut.edu.cn zhzhang.sdu@gmail.com.
Xiuzhen
X
Gao
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, People's Republic of China gaoxz@sdut.edu.cn zhzhang.sdu@gmail.com.
Zheng
Z
Zhang
School of Life Science, Shandong University of Technology, Zibo, People's Republic of China.
Ya'nan
Y
Zhang
School of Life Science, Shandong University of Technology, Zibo, People's Republic of China.
Ying
Y
Li
School of Life Science, Shandong University of Technology, Zibo, People's Republic of China.
Heng
H
Zhu
School of Life Science, Shandong University of Technology, Zibo, People's Republic of China.
Sheng
S
Wang
School of Life Science, Shandong University of Technology, Zibo, People's Republic of China.
Cun
C
Li
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000068536:Bacillota / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D007651:Keto Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D005810:Multigene Family; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007651:Keto Acids; D000594:Amino Acid Oxidoreductases; C020032:diaminopimelate dehydrogenase
d-amino acid; functional divergence; meso-diaminopimelate dehydrogenase; protein evolution; subfamily